Note: Descriptions are shown in the official language in which they were submitted.
HA135b
,.. .
This invention relates to new pro~line derivatives
and related compounds which have the general formula
(I) R
R~ IRl ~ H2C - (C~l)m
2 ~ )n ~H-~ CO - N - - ~H-COR
wherein R is hydroXy, NH2 or lower alkoxy;
: Rl and R4 each is hydrogen, lower alkyl,
phenyl or phenyl-lower alkyl;
R2 is hydrogen, lower alkyl, phenyI,
substituted phenyl wherein the phenyl
substituent is halo, lower alkyl or
lower alkoxy, phenyl-lower alkyl,
diphenyl-lower alkyl, triphenyl-lower
alkyl, lower alkylthiome~hyl, phenyl-
lower alkylthiomethyl, loMer alkano~l-
amidomethyl, R5-C-, R5-~-C-,R5-NH-C-,R6~S- or R7;
; R3 ls hydrogen,hydroxy or lower alkyl;
R5 is lower alkyl, phenyl o~ phenyl-lower
-1-
: '
- .
~ 4 ~IA135b
alkyl;
R6 is lower alkyl, phenyl, substituted
phenyl, (wherein the phenyl substituent
is halo, lower alkyl or lower alkoxy),
hydroxy-lower alkyl or amino(carboxy)~
lower alkyl;
13
(HC) - CH2 IRl R4
R7 is R-OC-HC - N - CO - CH--(CH)- S(O) ;
M is 0 or S;
m is 1 to 3;
n and p each is 0 to 2,
and to processes for maklng them.
The asterisks indicate asymmetric carbon atoms.
Each of the carbons bearing a substituent Rl, R3 and R~
is asymmetric when that suhstituent is~other than hydroyen.
~he invention in its broad aspects includes proline
and related derivatives having formula I above. Within this
broad group, because of their properties, certain subgroups -
are preferred over ot~ers.
Broadly preferred are those compounds of formula I
wherein R is hydroxy or lower alkoxy; Rl is hydrogen or
lower alkyl; R2 is hydrogen, R5-CO, R6-S-, or R7; R3;and
R~ each is hydrogen; R5 is lower alkyl, especially methyl
:: ~
or phenyl; X6 is lower alkyl, especially methyl or ethyl;
m is 2, n is 0, 1 or 2, especially 1; and R7 wherein R~ Rl,
,~:
R3, R~, m and n have the same preferences as above and p is 0.
-2-
Especially preferred are those compounds which have the
formula
(II)
R2 S (CH2)n - CH - CO~ N ~ COR
* *
wherein R is hydroxy or lower alkoxy;
Rl is hydrogen or lower alkyl; -
R2 is hydrogen, R5-CO-, R6-S- or R7;
R5 is lower alkyl or phenyl, especially the first;
R6 is lower alkyl; ~:
and n is 0, 1 or 2. ~.
Within the group of compounds represented by formula `~
II, the following are still more preferred subgroups in
the order (a to r~ of increasing preference to the com-
pounds which are especially preferred embodiments~
a) R is hydroxy
b) n is 1
:~ 20 c) R2 is hydrogen or lower alkanoyl
d) R2 is hydrogen
e) R2 is acetyl
f) Rl is hydrogen or lower alkyl .~
:''
g) Rl is hydrogen or methyl :
~ ~.
h) R is hydroxy, Rl is hydrogen or methyl
i~ R is hydroxy, Rl is hydrogen or methyl, R2 is hydro~
gen or acetyl and n is 0, 1 or 2 ~;
j) R is hydroxy, Rl and R2 each lS hydrogen and n`is~Q ~ .
.
, ;,
- 3 - :
: '
~ &9~ :
k) R is hydroxy, Rl is hydroyen, R2 is acetyl an~ n is
1~ R is hydroxy, Rl is methyl, R2 is acetyl and n is 1
m) R ls hydroxy, Rl and R2 each is hydrogen and n is 1
n) R is hydroxy, Rl is methyl, R2 is hydrogen and n is
o) R is hydroxy, Rl is hydrogen, R2 is lower alkylthio
and n is 1
p) R2 is
lo ~ 71
R-OC N - OC~-CH - (CH2)n-S~;
each R is hydroxy; Rl is hydrogen or lower alkyl, es-
pecially hydrogen or methyl; and n is 0 to 2, es-
pecially 1
M
q) R2 is R5-M-C- wherein M is O or S
M
r) R2 is R5-NH-C- wherein M is O or S, preferably S.
It will be appreciated that combinations of the fore-
going, where applicable, are amang the preferred groups. ;~
The stereoisomers in which the proline is in the L-
form are especially preferred.
The lower alkyl groups represented by any of the vari-
ables include straight and branched chain hydrocarbon
radlcals ~rom methyl to heptyl, for example, methyl, ethyl, ~
propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, iso- ~;
pentyl and the like. The lower alkoxy groups are or the
same kind having 1 to 7 carbons linked to oxygen, for ex-
ample, methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-
butoxy, t-butoxy and the like. The Cl-C4 members, espe-
cially Cl and C2 members, of both
~ - 4 -
,
~186~ HA135b
types are preferred. Phenylmethyl is the preferred phenyl-
lower alkyl group.
The lower alkanoyl groups are those having the acyl
radicals of ~he lower (C2-C7) fa-tty acids, Eor example,
acetyl, propionyl, bu-tyryl, isobutyryl and the like. Similarly,
those lower alkanoyl groups having up to four carbons, and
especially acetyl, are preferred.
The four common halogens are included by the term
'!halo" but chlorine and bromine are preferred. The substi-
tuted phenyl groups preferably bear the substituent in the
4-position of the ring. The hydroxy-lower alkyl groups
have a hydroxy group on an alkyl chain like those described
above, preferably on the terminal carbon, e.g., hydroxymethyl,
2-hydroxyethyl, etc. The amino(carboxy)lower alkyl groups
have one amino and one carboxy on a lower alkyl group such
as those described above, preferably both on one carbon,
e.g., on the terminal carbon as in the preferred 2-amino-2- ~ ~ -
carboxyethyl group.
The products of formula I and the preferred subgroups
can be produced by various methods of synthesis.
In general, the products of this inven~ion are
produced by acylating a compound of the formula
(III) 3
I '
H2C ( ICH)m
HN CH - COR
with an acid of the formula
(IV) R~ 11
R2-- S - (CH) - CH - COOH
--5--
~ 4 HA135b
or its chemical equivalent.
Thus, the inal product can be produced not only by direct
acylation with an acid of formula IV but also by intermediates
such as (a~ ~-haloalkanoic acids of the formula
R4
(V)
X -(CH)n CH - COOH ~
wherein X is bromo, chloro or iodo, or (b~ a tosyloxyalkanoic
acid, i.e., X ln formula V is -tosyloxy (CH3 ~ SO2Q-)
(c) a substituted acrylic acid of the formula
14 1l
(VI) CH C - COOH
The product of this acylation is then subjected to displacement
or addition with the anion of a thiol or thioacid of the
formula
(VII) R2 - SH
Acylation can also be effected with a thiolactone of the
formula
R4
(VIII) (CIH)n - CH
S =O
wherein n is 1 or 2, or a mercaptoalkanoic acid of the formula
(IX) Y - S (CH)n - CH COR
:
-6-
~ 4 ~A~35b
wherein Y is R2 or, in addition, if a product of formula I
wherein R2 is hydrogen is desired, then Y can also be a
protecting group such as (a) CH3~ C112-, (b) ~ ,
(c) CH3CONHCH2, (d) R~O-C-CH-(CH) -S- or other sulfur
protecting group. 'IDeprotection" can be effected by
conventional means such as treatment with hot trifluoroacetic
acid, cold trifluoromethanesulfonic acid, mercuric acetate,
sodium in liquid ammonia, zinc and hydrochloric acid or the
like~ For a review of these methods see Me-thoden der
Organischen Chemie (Houben-Weyl), Vol. XV, part I, page
736 et seq. (1974).
When the acid of formula IV is used as the acylating
agent, the acylation can be efected in the presence of a
coupling agent like dicyclohexycarbodiimide or the like,
or the acid can be activated by formation of its mixed
anhydride, symmetrical anhydride, acid chloride, acid
ester or use of Woodward reagen-t K, N-ethoxycarbonyl-2-
ethoxy-1,2-dihydroquinoline or the like. For a review of the
methods for acylation, see Methoden der Organischen Chemie
(Houben-Weyl), Vol. XV, part II, page 1 et seq. (1974).
Compounds of formula III lnclude, for example,
proline, hydroxyproline, 4-methylproline, plpecolic acid,
5-hydroxypipecolic acid, azetidine-2-carboxylic acid, their
lower alkyl esters and the like. The acylation of such
compounds is described in greater detail below.
According to a preferred method for producing compounds
;~ of formula I, especially wherein R2 is R5-CO~, an acid or
ester of formula III is coupled with a haloalkanoic acid of
the formula
-7-
HAl35b
(V) R4
X-(CH) - CH- COOH
wherein X is a halogen, preferably chlorine or bromine. This
can be effected by one of the known procedures in which the acid
IV is activated, prior to reaction with the acid III, involving
formation of a mixed anhydrlde, symmetrical anhydrlde, acid
chloride, active ester, or use of Woodward reagent K, EEDQ
(N-ethoxycarbonyl-2-ethoxy-l,2-dihydroxyquinoline) or the
like.
The product of this reaction is a compound of the
formula
(X) 13
4 1l H2C (CH)m
: X - (CEI) - ca - co - N - CH-COR
The product o formula X is subjected to a displacement ~ :
reaction with the anion of a thioacid of the formula
(VII)
R2-5H
yielding a product of the formula
(XI)
R
~ R4 : Rl~ H2C1 (1 )m
R2- S (CH) - CH - CO - N - CH-COR
When R2 is R5CO, this product can then be converted to
the product . I - ::
(XII) R
4 1l 21 (1 )m
HS - (CH)n- CEI - CO - N -CH-COR
HA135b
~ ,
by ammonolysis. When R2 is a protecting group, then -the
compound of formula XII can be obtained by"deprotection"
as described above. When R is an ester group (i.e., R
is lower alkoxy), the ester group can be removed, e.g.,
when R is tert. butoxy or tert. amyloxy, by treatment of
the ester of formula XI or XII with trifluoroacetic acid
and anisole to give the corresponding free acid. When other
alkoxy groups are present, alkaline hydrolysis will yield
the corresponding acid.
A variation of this procedure involves the use of
an acrylic acid of the formula
(VI) 14 ~
CH=C-COOH
as starting material. This acrylic acid is first converted to
the acid halide form then made to react with a compound of
formula III to obtain a compound of the formula
(XIII) R3
R~ Rl H2C (IC )m
CH=C - CO- N - CH-COR
and this intermediate is subjected to the addition reaction
with the thiol or thioacid VII as described above.
A tosyloxyalkanoic acid of the formula
(XIV)
R4 IRl
CH3- ~ - 9O2O-(CH)n- CH- COOH
can also be used as the agent to acylate the acid of formula
III, then the acylation product lS subjected to the displace-
ment reaction, etc., as descrihed above.
The acrylic acid of formula VI can alternatively be
first made to react with the thioacid of forrnula VII to
IIA135b
obtain a product of the formula
(XV)
R~ R
R2-S-CH-CH-COOH
which is converted to its acid halide, e.g., with thionyl
chloride, then coupled to the compound of formula III and
the same sequence as above then followed.
The acid or ester of formula III can also be acylated
with a "protected" form of a ~-mercaptoalkanoic acid of khe
formula
(~VI) 14
R~-S-(CH~ -CH-COOH
wherein R8 i5 the "protec-ting" group. Such "protecting" yroups
can take the form described above.
Following the acylation, the produck can be "deprotected"
by one fo the known methods referred ko above.
SkiIl another acylating agent can take khe form of a
thlolac-tone, e.g., ~-propiothiolactone, a-mekhyl-~-propiothio-
lactone or khe like.
Additional dekails of preferred modes of producing
compounds of this invention can be found in the followin~
and in khe specific examples.
According ko a particularly preferred modification,
the acid or ester of formula III is acylated with a halo-
alkanoyl halide of the formula
(XVII) R~ Rl
X-~CH)n- CH-COX
wherein each X is independenkly a halogen, preferably chlorine
or hromine, Rl is hydrog~n, lower alkyl or phenyl-lower alkyl
-~.n-
HA135b
and n is 0, 1 or 2. This reaction is effec-ted in an alXallne
medium, e.g., dilute alkali metal hydroxide solu-tion, alkali
metal bicarbona-te or alkall metal carbonate solution at a
reduced temperature, e.g., about 0 to 153C. The reaction
product is subjec-ted to displacement with the anion of the
thiol or thio acid of the formula VII above, also in alkaline
medium, preferably alkali metal carbonate solution, and then
worked up in conventional manner. The product of this
reaction, wherein R2 of formula I is R5-CO, is converted to
the product wherein R2 is hydrogen by ammonolysis, e.g.,
alcoholic ammonia or concentrated ammonium hydroxide solution,
or alkaline hydrolysis, e.g., with aqueous metal hydroxide.
When an acid of formula III is used as starting material, the
final product obtained as the free carboxylic acid can then be
:,
converted to its ester, for example by esterification wlth
a diazoalkane, like diazornethane, l-alkyl-3-p-tolyl-triazene,
like l-n-butyl-3-p-tolyltriazene or the like. Treatment of an
ester, preferably the methyl ester, with an alcoholic ammonia
:
solution, converts the free acid to the amide, i.e., R is NH2.
According to another variation, an ester, preferably
the t-butyl ester, of formula III, in an anhydrous medium
such as dichloromethane, tetrahydrofuran, dioxane or the like,
is treated with a thioalkanoic acid of the formula
(XVIII)
2 2 n
in the presence of dicyclohexylcarbodiimide, N,N'-ca~bonyl-
bisimidazole, ethoxyacetylene, diphenylphosphoryl azide or
similar coupling agents at a temperature in the range of about
0 to 10 C. The ester group (R) can then be removed, for
--11--
HA135b
example, by treatment with trifluoroacetic acid and anisole
at about room temperature.
When an ester of formula III (e.g., R is lower
alkoxy, especially~ t-butoxy) is acylated with a thiolactone,
e.g., ~-propiothiolactone, ~-methyl-~-propiothiolactone or the
like, khe reaction can be effected in an anhydrous solvent
like tetrahydrofuran, dioxane, methylene ch:loride or the
like at about 0 C. to about room tempera~ure. The ester
group can be removed wit~ anisole and trifluoroacetic acid
as described above. M
In similar manner, when R2 is R5-M-C-,-~products of
formula I having this substituent are formed by reacting a
compound of formula XII with the halogenated compound
(XIX) M
R5-M-C-X
or alternatively reacting a compound of formula X with an ~ ;
alkali metal salt or alkaline earth metal salt of the
formula
(XX) M
R5-M-C-S-Me
wherein Me represents the alkali metal or alkaline earth metal.
M
When R2 is R5-NH-C-, products of formula I having this
substituent are produced by reacting a compound of formula XII
with the appropriately substituted isocyanate or isothio-
cyanate of the formula
(XXI)
R5-N=C=M - `
Alkernatively, the same products can be produced by coupling
an acid of the formula
3~
-12-
- :
IIAl 3 5h
( XXII )
M R R
Il 14 11
R -NH- C S- (CH)-- CH-COOH
with an amino acid of formula III.
Compounds of formula I, wherein R2 :is lower alkyl, phenyl,
substituted phenyl, phenyl-lower alkyl, tr:iphenyl-lower alkyl,
lower alkylthiomethyl or phenyl-lower alkyLthiomethyl are
produced by reacting a compound of formula XII with the
corresponding halide R2X or by reacting a compound of formula X
with the corresponding thiol ~2SH in the same manner as
described above.
When R is lower alkanoylamidomethyl, the product of
formula I is produced by condensing a compound of formula XII
with the corresponding hydroxymethyl-lower alkanoylamide of
th formula
(XXIII) lower alkyl-CO-NHCH2OH
in thè presence of an acid catalyst like trifluoroaoetic acid.
Products of formula I wherein R2 is R6-5 can be
prepared by any of the known methods for the synthesis of
mixed disulfides, e.g., by the reaction of a compound of
formula(XII)with a thiosulfinate(xxIv), thiosulfonate (XXV~,
su]fenyl halide (XX~I), thiosulfate (XXVII) or sulfenyl
thiocyanate (XXVIII) O
(XXIV) ~R6-S~S-R6 , (XXV) R6 11 5 R6 ' 6
(XXVII) R6-$-SO3H, (XXVIII) R6-S-SCN
In the particular case wherein R7 is
R
l4 R~ CH2 - (lH)n
-S(O) - (CH) -~ CH - CO - N ~ CH-COR,
p n
HA135b
R, Rl, R3 and R~ are the same as -the corresponding subs-ti-tuents
in formula I and p is O, the symmetrical disulfides can be
obtained by direct oxidation of a compound of formula XII
with iodine. When p is 1 or 2, such products are obtained
by the stepwise oxidation of the corresponding compound wherein
p is O. Mixed disulfides are obtained by the modifiGation
shown in the examples.
Products of formula I have one or more asymmetric carbons.
When Rl, R3 or R4 is other than hydrogen the carbon to which
it is attached is asymmetric. These carbon atoms a~e
indicated by an asterisk in formula I. The compounds
accordingly exist in stereoisomeric forms or in racemic
mixtures thereof. Al:L of these are within the scope of
the invention. The above described synthesis can utilize
the racemate or one of the enar2tiomers as starting material.
When the racemic starting material is used in the synthetic
procedure~ the stereoisomers obtained in the product can be
separated by conventional chromatographic or fractional
crystallization methods. In general, the L-isomer wi~h
respect to the carbon of the amino acid constitutes the
preferred isomeric form. Also the D-isomer with respect to
the a-carbon in the acyl side chain (i.e., the carbon
bearing Rl) is preferred.
~ The compounds of this invention orm basic salts
with various inorganic and organis bases which are also
within the scope af the invention. Such salts include
ammonium salts, alkali metal salts like sodium and potassium
salts (which axe pre~erred~, alkaline earth metal salts like
the calcium and magnesium salts, salts with organic bases,
e.g., dicyclohexylamine salt, benzathine, N-methyl-D-glucamine,
HA135b
hydrabamine salts, salts with amino acids like arginine,
lysine and the like. The non-toxic, physiologically acceptable
salts are preerred, although other salts are also useful,
e.g., in isolating or purifying the product, as illustrated
in the examples in the case O,c the dicyclohexylamine salt.
The salts are ormed in conventional manner by reacting
the free acid form of the product with one or more equivalents
of the appropriate base providing the desired cation in a
solvent or medium in which the salt is insoluble, or in
water and removing the water by freeze drying. By neutralizing
the salt with an insoluble acid like a cation exchange resin
in the hydrogen orm (e.g., polystyrene sulfonic acid resin
like Dowex 50) or with an aqueous acid and extraction with~an
organic solvent, e.g., ethyl acetate, dichloromethane or the
like, the free acid form can be obtained, and, if desired,
another salt formed.
,
:
_ :
'
15-
.. ,
. .
HA135b
Additional experimental details are Eound in the
examples which are preferred embodiments ancl also serve as
models for the preparation of other members oE the group.
The compounds of this invention inhi bit the conversion
of the decapeptide angiotens in I to angiotensin II and
therefore are useful in reducing or relieving angiotensin related
hypertension. The action of the enzyme renin Oll angiotensinogen,
a pseudoglobulin in blood plasma, produces angiotensin I.
Angiotensin I is converted by anyiotensin converting enzyme
(ACE) to angiotensin II. The latter is an active pressor
substance which has been implicated as the causative
agent in various forms of hypertension in various mammalian
species , e . g ., rats and doys . The coM~ounds o~ t}lis invention
intervene in the angiotensin (renin)-tanyiotensin I ~angiotensin II
se~uence by inhibiting anglotensin conver-ting enzyme and reducing
or eliminating -the formation of the pressor substance
angiotensin II . Thus by the adminis tration of a con~position
contalning one or a cornbination o f compounds oE formula I
or physiologically acceptable salt thereof, angiotensin
dependent hypertension in the species of marnmal suf feriny
therefrom is alleviated. A slngle dose, or pre~erably two
to four divided daily doses, provided on a basis of about 0. 1 to
100 mg. per kilogram per day, preferably about 1 to 50 mg. per
kilogram per day is appropriate to reduce blood pressure as
indicated in the animal model expeximents described by
S.L~ Engel, T. R. Schaeffer, M.H. Waugh and B. Rubin,
Proc. Soc. Exp. Biol. Med. 143 j 483 (1973) . The substance
is preferably aclministered orally, but parenteral routes
such as subcutaneous, intramuscular , intravenous or
-16-
HA135b
intraperitoneal can also be emyloyed.
Tiie compounds of this invention can be utilized to
achieve the reduction o~ blood pressure by formulating in
compositions such as tablets, capsules or elixirs for oral
administration or in sterile solutions or suspensions for
parenteral administration. ~bou-t 10 to 500 my. of a
- compound or mixture of compounds oE formula I or physiologically
acceptable salt is compounded with a physiologically
acceptable vehicle, carrier, excipient, binder, preservative,
s~abilizer, flavor, etc., in a unit dosage foxm as called
for by accepted pharmaceutical practice. The amount of active
substance in these compositions or preparations is sucl- tha~
a suitable dosaye in the range indi.cated is obtained.
Illustrative of the adjuvants which may be incorporated
in t~blets, capsules and the like are the followin~: a binder
such as gum tragacanth, acacia, corn starch or gelatin; an
excipient such as dicalcium phosphate; a disintegra~ing agent
such as corn starch, potato starch, alginic acid and the like;
a lubricant such as magnesium stearate; a sweeteniny ayent
such as sucrose, lactose or saccharin; a flavoriny agent such
as peppermint, oil of wintergreen or cherry. When the dosaye
unlt form is a capsule, it may contain in addition ~o Inaterials
of the above type a liquid carrier such as a fa~ty oiI. Various
other materials may be present as coatinys or to o-therwise
modify the physical form of the dosage unit. For instance,
tablets may be coated with shellac, suyar or both. A syrup
or elixir may contain the active compound, sucrose as a
sweeteniny ayent, methyl and propyl parabens as preservatives,
a dye and a flavoriny such as cherry or orange flavor.
Sterile coml~ositions ~or in~ectiol- can ~e ~ormula~ecl
-17-
~ 4 ~IAl35b
accordiny to col-ventional pllarmclceutical practice by
dissolving or suspending the active. substance in a vellicle
such as water Eor injection, a naturally occurriny vegetable
oil like sesame oil, coconut oil, peanut oil, cottonseed
oil, etc., or a synthetic fatty vehicle like ethyl oleate or
the like. Buffers, preservatives, an~ioxidants and tlle like
can be incorporated as required. ,
The following e~amples are illustrative of the invention
and constitute especially preferred embodiments. All tempera-
tures are in degrees celsius.
- -18-
8~
HA13$b
E:xam~:l.e 1
.
1-(2-~3enzoyl~ ioacctyl)-L-Proline
L-Proline (5.75 g.) is dissolved in N sodium hydroxide
(50 ml.) and the solu-tion is chilled in an ice-water bath.
Sodium hydroxide 2N (26 ml.) and chloroacetyl chloride (5.65 g.)
are added andthe mixture is s-tirred vigorously at room
temperature for three hours. ~ suspension of thiobenzoic acid
(7.5 g.) and potassium carbonate (4.8 g. ) in water (50 ml.
is added. After 18 hours stirring at room -temperature, the
reaction mixture is acidified and extrac-ted with ethyl acetate.
The ethyl acetate layer is washed with water, dried over
magnesium sulfate and concentrated to dryness in vacuo. ~he
residue (14.6 g.) is dissolved in ethyl acetate (lS0 ml.) and
dicyclohexylamine (lL ml.) is added. The crystals are filtered
and recrystallized from ethyl acetate, yield 5.7 g. m.p.
151-152 . To convert tlle salt to the acid, the crystals are
dissolved in a mixture of 5~ -aqueous potassium bisulfate
(100 ml.) and ethyl acetate (300 mlr). The oryanic phase is ;~
washed once with water, dried over magnesiuln sulfate and
concentrated to dryness in vacuo, yield 3.45 g.
Example 2
1-(2-Mercaptoacetyl)-L-Proline
1-(2~Benzoylthioacetyl)-L~proline (3.4 g.) is dissolved
in a mixture of water (10.5 ml.) and concentrated al~nonia (6.4 ml.)~
After one hour, the reaction mixture is diluted with water
and filtered. The filtrate is extracted witSI ethyl acetate
and then acidi~ied with concentrated hydrochloric acid,
saturated with sodium chloride and extracted twice with ethyl
acetate. The ethyl acetate extracts are wasl~ed with saturated
sodium chloride and concentrated to dryness, yield 1.5 c
~19-
;4
The product, 1-(2-mercaptoacetyl)-L-prol:ine is crystallized
from ethyl ace-tate (m.p. 133-13S).
Example 3
c ~ roline Methyl Ester
___
1-(2-Benzoylthioacetyl)-L-proline obtained in Example
1, is dissolved in methanol and an ethereal solution of
diazomethane is added until there is a persistent yellow
color. After 15 minu-tes, a few drops of acetic acid are
added and the solvent is removed in vacuo to obtain 1-(2-
benzoylthioacetyl)-L-proline methyl ester.
Example 4
~ -
1-(2-~ercaptoacetyl)-L-Proline Amide
The product of Example 3 is dissolved in 10% metha-
nolic ammonia and the solution is s-tored at room tempera-
ture in a pressure bottle. When thin layer chromatogra-
phic analysis indicates that the two es-ter functions have
been ammonolyzed, the reaction mixture is concentrated to
dryness to obtain l-(2-mercaptoacetyl)-L-proline amide.
Example _
1-(2-Benzoylthioacetyl)-L-Hydroxyproline
By substituting L-hydroxyproline for the L-proline in
the procedure of Example 1, 1-(2-benzoylthioacetyl)-L-
hydroxyproline is obtained.
Example 6
1-(2-Mercaptoacetyl)~L-Hydroxyproline
By treating the product of Example 5 with ammonia as
in Example 2, 1-(2-mercaptoacetyl)-L-hydroxyproline is
obtained.
Example 7
1-(2-Benzoylthioacetyl)-L-Azetidine-2-Carboxylic Acid
By substituting L-aze-tidine-2-carboxylic acid for the
- 20 -
~'
~ A13S~
L-proline in the ~rocedure oE ~xam~le 1, 1-(2-benzoyl-
thioacetyl)-L-azetidine-2-car~oxylic acid is obtained.
~ ~,
1-~2-Mercaptoacetyl)-L-~zetidine-2-Car~oxylic ~cid
By treating the product of ~xample 7 with ammonia
as in Example 2, 1-(2-mercaptoacetyl)-L-azetidine-2 carboxylic
acid is obtained.
Example 9
1-(2-B _ oylthioacetyl)-L-pipecolic Acid
By substitutinc~ L-pipecolic acid for the L-proline
in the procedure of Example 1, 1-(2-benzoylthioacetyl)-L-
pipecolic acid is obtained.
~xample 10
1 (2-Mercaptoacetyl)-L-Pipecolic Acid
By treating the produc-t of Example 9 with ammonia as
in Example 2, 1-(2-mercaptoacetyl)-L-pipecolic acicl is
obtained.
xample 11
1-(2-~enzoyltlliopropanoylj-L-Proline
L-Proline (5.75 g. ) is dissolved in aqueo~s N sodium
hydroxide (50 ml.) and the solution is chilled in an ice
bath with stirring. 2N sodium hydroxide (25 ml.) ancl 2-bromo-
propionyl chloride (8~57 g.) are added in that order and
the mixture is removed from the ice bath and stirred at room
tempexature for one hour. A mixture of thiobenzoic acid
(7.5 g.) and potassium carbonate (4.8 g.) in water (S0 ml.)
is added and the mixture is stirred overnight at rQom
temperature. ~fter acidification witll concen~trated hydro-
chloric acid, the aqueous solution is extracted with e~hyl
acetate and the organic phase is washed with water, dricd and
-21-
- . ~ ~' . . , '
B64
HA135b
concentrated to dryness. The resiclue (14.7 ~.) is chromatographed
on a column of 440 g. o silica gel witll a mlxture of
benzene-acetic acid (7:1~. The ractions containinc3 the
desired material are pooled, concentrated ~o dryness, and the
residue is preci~itated -twice with ether-hexane and converted
~o a dicyclohexylamine salt in ether-hexane, yield 9.4 g.
m.P.l(142) 148-156.The dicyclohexylamine salt is converted back to
the acid as in Example 1, yield 5.7 g
xample 12
1-(2-Mercaptopropanoyl)-L-Proline
1-(2-~enzoylthiopropanoyl)-L-proline (5.7 g.) is
dissolved in a mixture o water (12 ml.) and concentrated
ammonium hydroxide (9 ml.) with stirring. ~ter one hour,
the mixture is diluted with water (lQ ml.) and iltered.
The filtrate is extracted twice with ethyl acetate, concentrated
to one-third of the original volume, acidi~ied with concentrated
hydrochloric acid and extracted with ethyl acetate. The
organic phase is washed with saturated sodium chloride,
dried and concentrated to dryness in vacuo. The residue,
1-(2-mercaptopropanoyl)-L-proline, is crystallized from
eth~l acetate-hexane, yield 3 g.,m.p. (105) 116-120 .
~xample 13
1-(3'-Benzoylt:hioproL~anoyl)-L-Proline
L-Proline (5.75 g.) is dissolved in normal sodium
hydroxide (50 ml.) and the solution is chilled in an ice bath.
3-Bromopropionyl chloride (8.5 g.) and 2N sodium hydroxide
(27 M1.) are added and the mixture is s~irred for 1-0 m1nutes
in the ice bath and three hours at room temperaturc. A
suspension of ~hioL~en~oic aci.d (7.5 q.) antl potassium carbonate
(4.5 g.) in water ( 50 ml.) is added antl the mixture is stirred
c
-22-
1864 ilA1~ 5b
for 18 hours a~ room ~em~erature. Af~er acicliEication witl
concentrated hydrochloric acid, the aqueous ~hase is
extraete~ twice with etllyl acetate. The organic layers
are driecl over magnesium sulfate and concentrated to dryness
in vacuo -to obtain 1-(3-benzoyltlliopropanoyl)-L-proline,
yield 7.1 g;, m.p. 101 102 ~etilyl acetate-hexane).
Example 1
L-Proline tert.-butyl ester
,
I.-Proline (230 g.) is dissolved in a mixture of
wa-ter (1 1.) and 5 N sodium hydroxide (400 ml.). The solution
is chilled in an ice bath, and under vigorous stirring, 5 N
sodium hydroxide (460 ml.) and benzyloxycarbonyl chloride
(340 ml.) are added in five equal aliquo~s during a half
hour period. ~fter one l~our stirring at room temperature, the
mixture is extracted twice with ether and acidified with
concentrated hydroc}lloric acid. The precipitate is ~iltered
and dried. Yield 442 g. m.p. 78-80.
Tlle benzyloxycarbonyl-L-proline thus obtained
(l~O g.) is dissolved in a mixture of dichlorometllane
(300 ml.),liquid isobutylene (800 ml.) and concelltratecl
sulfurie acid (7.2 ml.). The solution is shaken in a
pressure bot-tle ~or 72 hours. The pressure is released,
the isobutylene is allowed to evaporate and the solution is
washed with 5% sodium carbonate, water, dried ovex mac3nesium
sulfate and concentrated to dryness in vacuo,to obtain benzyloxy-
carbonyl-L-proline tert.butyl ester, yield 205 g.
Benzyloxycarbonyl-L-proline tert.butyl este~ (205 g.)
is dissolved in absoIute ethanol (1~2 1) and hydrogenatecl at
normal pressuxe with 10% Pd on carbon (10 g.) until only a
-trace of carbon dioxide is observed in the hydrogen exit cJas
-23-
:
i5i6~
HA135b
(24 hours). The catalyst is Eiltered off arld the fil~rate
is concentrated ir. vacuo at 30 mm. ~IcJ. The residue is
.
distilled in vacuo,,to obtain L-proline tert.butyl ester,
b.p.lmm 50-51
Exampl~ 15
1-(3-Acetylthiopropanoyl)-L Proline tert-butyl Ester
L-Proline tert-butyl ester (5.13 g.) is dissolved in
dlchloromethane (40 ml.) and the solution is chilled in an
ice-water bath. A solution of dicyclohexylcarbodiimide
(6.18 g.) in dichloromethane (20 ml.) is added followed
immediately by 3-acetylthiopropionic acid (4.45 g.). A~-ter
15 minutes stirring in the ice-water bath and 16 hours at
room tempera-ture, the precipitate is filtered off and the
filtrate is concentrated to dryness in vacuo. l`he residue is
,
dissolved in ethyl acetate and washed neutral. Th~ orr3anic
layer is dried over magnesium sulfate and concentrated to
dryness in vacuo to obtain 9.8 cJ. of 1-(3-acetylthiopropanoyl)-L-
proline tert-butyl ester~
Example lG
1-(3-Acetylthi~propanoyl~-L-Proline
1-(3-Acetylthiopropanoyl)-L-proline-t-buty1 ester
(4~7 g.) is dissolved in a mixture o anisole (34 ml.) and
trlfluoroacetic acid (68 ml.) and the mixture is kept at
room temperature for one hour. rrhe solvents are removed
in vacuo and the residue is precipitated from ether-hexane
several times. The residue (3.5 g.) is dissolved in
acetonitrile (25 mlO) and dicyclollexylamine (2.8 ml.) is
added. The crystalline salt is filtered and recrystallized
from isopropanol. Yield 3.8 y.. mOp~ 176-177 . 'l`he salt
is reconverted -to 1-(3-acetylthiopropanoyl)-L-proline as in
-~4
HA135b
Example 1, yield 1.25 g., m.p. 89-90 ~ethyl acetate-hexane).
Examplc 17
1-(3-Mercaptopropalloyl)-L-proline ter~-bu~yl ~ster
To a solution of L-proline -tert-butyl ester (`3.42 g )
in dry tetrahydrofuran (10 ml.) chilled in an ice bath,
propiothiolactone (1.76 9.) is added. ~fter 5 mirlutes storage in
- the ice bath and three hours at room temperature, the reaction
mixture is diluted with ethyl acetate (200 ml.) and washed
with 5~ potassium bisulfate, and water. The organlc layer
is dried over magnesium sulfate and concentrated~to dryness in
vacuo. The residue 1-(3-mercaptopropanoyl)-L-~proline tert-
;,
butyl ester is crystallized from ether-hexane, yield 3.7 g.,
m.p. 57 58.
'
Example 18
1-(3-Mercaptopropanoyl)~-L-Proline
Procedure ~
1-13-Benzoy~lthiopropanoyl)-L-proline (4.9 g.~ls
dissolved in a mixture of water~(8 ml.) and concentrated ammonium
hydroxide (5.6 ml.) and the solution is stored with stirring
under argon for one hour~ The reaction mixture is diluted
with water, filtered, and the filtrate is extracted with
ethyl acetate~ The aqueous phase is acidified with concentrated
hydrochloric acid, saturated with sodium chloride and extracted
with ethyl acetate. The organic la~ers are washed with
saturated sodium chloride, dried over magnesium sulfate, and
~concentrated to dryness in vacuo. The residue,l-~3-mercapto-
propanoyl)-L-proline, is crystallized from ethyl acetate
hexane, yield 2.5 y., m.p. 63-70 .
Procedure B
1-(3-~cetylthiopropanoyl)-L-proline (0.~ 9.) is dissolved
- -25-
,
HA135b
in 5.5 N methanolic ammollia (5 ml.) and the solu~ion ke~
under ar~on at roorn temperature. ~Eter 2 hours th~ solvent is
removed in vacuo, the residue is dissolved in water and applied
to an ion exchange column on the H cyclelDowex 50~Analytical
grade)]and eluted with water. The fractiolls that ~ive thiol
positive reaction are pooled and concentrated to dryness,
yield 0.6 g. This product is crystallized from ethyl acetate-
hexane as in Procedure A to obtain 1-(3-mercap-to~ropanoyl)-L-
proline.
Procedure C
1-(3-Mercaptopropanoyl)-L-proline t-butyl ester (2.3 g.)
is dissolved in a mixture of anisole (20 ml.) and triEluoro-
acetic acid (45 ml.). After one hour storage at room temperature
under aryon, the reaction mixture is concentrated to dryness
in vacuo and the residue precipitated from ethyl acetate-
hexane several times. The residue (1.9 cJ.) is dissolved in
ethyl acetate (30 ml.) and dicyclohexylamine (1.85 ml.) lS
added. The crystalline salt is filtered and recrystallized
from isopropanol, yield 2 g. m.p. 187-188.
The salt is converted to the acid as in Example 1,
yield 1.3 g. The product is crystallized from etl-yl acetate
hexane as in Procedure A.
Salts
Sodium
1-(3-Mercaptopropanoyl)-L-proline (500 mg.) is dissolved
in a mixture of water ~2~5 m~.) and N sodium hydroxidc (~.S ml.).
The solution is freeze dried to obtain the sodium s~lt.
Magnesium
1-(3-Mercaptopropanoyl)-L-proline (500 mg. ?,
maynesium oxide (49~5 mg.), and water ~10 ml.) are stirred
-26-
HA135b
with slic3ht heating until complete solution is obtailled.
Tllen the solvent is rernoved by freeze dryin~J -to obtain the
magnesium salt.
Calcium
__
1-(3~Mercaptopropanoyl~-2-proline t500 mg.) is
dissolved in a mixture o~ calcium l~ydroxide (91 mg.) and
water (10 ml.), and the solution is free~e dried to o~tain
the calcium salt.
Potassium
1-(3-Mercaptopropanoyl)-L-proline(500 mg.) is
dissolved in a mixture of potassium bicarbonate (246 mg.)
and water (10 ml.) and freeze dried to obtain the potassium
salt.
N-Methyl-D-Glucamine
1-(3-Mercaptopropanoyl)-L-proline (500 m~.) and
N-rnethyl-D-glucamine (480 mg.) are dissolved in water (10 ml.)
and freeze dricd to obtain the ~-methyl~D-glucamine salt.
~xample 19
1-(3-Mercaptopropanoyl)-L-IIydroxyproline
By substitutiny L-hydroxyproline for the L-proline in
the procedure of ~xarnple 11 and then -treating the product
by Procedure A of Example 18, 1-(3-benzoylthiopropanoyl)-L-
hydroxyproline and 1-(3-mercaptopropanoyl)-L-hydroxyproline,
respectively, are obtained.
~xample 20 ,~
1-(3-Mercaptopropanoyl)-L-Aze-tidine-2-CarboxYlic ~cid
By substituting L-azetidine-2-carboxylic acid tert-
: .
butyl ester (prepared by substituting L-azetidine-2- ~ -
carbox~lic acid for the proline in ~x~rnple 14) for ~lle L-
proline tert-butyl ester in the procedur.c ~f ~xaml~le L5,
. .
-27-
HA135b
treating the product as in ~xample 16 and tlle 1-(3-acetyl-
thiopropanoyl)-L-azetidine~2-carboxylic acid thus obtained by
Procedure B of Example 18, 1-(3-acetylthiopropalloyl)-L-
azetidine-2-carboxylic acid ter-t-butyl ester and 1-(3-mercapto-
propanoyl)-L-azetidine-2-carboxylic acid, respectively,
are obtained.
Example 21
1-(3-Mercaptopropanoyl~-L-Pipecolic ~cid
By substituting L-pipecolic acid tert-butyl ester
(prepared by substituting L-pipecolic acid ~or the L-proline
in Example 14) for the L-proline -tert-butyl ester in the
procedure of Example 15 and ~reatiny the product by Procedure C
of ~xample 18, 1-(3-mercaptopropanoyl)-L-pipecolic acid tert-
butyl ester and 1-(3-mercaptopropanoyl)-L-pi~)ecolic acid,
respectively, are obtained.
Example 22
1-(3-Mercaptopropanoyl)-~-Methyl-L-Proline
By substituting 4-methyl-L-proline for L-proline
in the procedure of Example :L3 and ~hen treating the product
by Procedure A of Example 18, 1-(3-benzoylthiopropanoyl)-
4-methyl-L-proline and 1-(3-mercaptopropanoyl)-4-metl~yl-L-
proline, are obtained.
~ xarnple 23
1-(3-Mercaptopropanoyl)-5-llydroxy-L-Piuecolic Acid
By substitutiny 5-hydroxy~ -pipecolic acid for L-
proline in the procedure of ~xample 13 and then trea-ting the
product by the Procedure A of Example 18, 1-(3-ben;zoyl-
thiopropanoyl)-S-hydroxy-L-pipecolic, and 1-(3-mercapto-
propanoyl)-5-hydroxy-L-pipecolic acid are ob~ained.
-28-
HA135
Example 2~
1-(3-Mercaptol~ropanoyl.)-D-Proline
By substituting D-proline ~or L-pYoline in the proc~dure
of Example 13 and then trea-ting the product by Procedure A of
Example 18, 1-(3-benzoylthiopropanoyl)-D-proline and 1-(3-
mercaptopropanoyl)-D-proline, m.p. 68-70 , are obtained.
- Example 25
3-Acetylthio-2-Methylpropanoic Acid
~ mixture of thloacetic acld~ ~50 y.) and metllacrylic
acid (40.7 g.) is heated on the steam bath for one hour and ' '~
then stored a-t room temperature for 18 hours. After confirming
by nmr spectroscopy that con~plete reaction of tlle metllacrylic
acid has been,achieved, the reaction mi,xture is distilled
in vacuo and the desired 3-acetylthio-2-methylpropanoic acid
is separated in the fraction with bolling point 128.5 -131
' (2.6 mmHc3.), yield 64 y. ,
Example 26 i~
3-Benzoylthio-2-Methylpropanoic Acid ~,
By substituting thiobenzoic acid for the thioacetlc
acid in the procedure of Example~2s, 3-benzoylthio-2-
methylpropanoic acid is obtained.
Example 27
3-Phenylacetylthio-2-MethylproE~anoic ~cid
By substituting thiophenylacetic acid for the
thioacetic acld in the procedure of Example 25, 3-phenyl-
acetyl thio-2-methylpropanoic acicd is obtained.
~.~ample 28
1-(3-~cetylthio-2-methylprc)E)anoyl)-L-Prolille tert-butyl Ester
L-Proline tert-butyl ester (S.l g.) i~ dissolved in
dichloro~ethalle (40 ml.) and the solution stirred and chilled
.
-29- ~
~Q~
~ 135b
in an ice bat}l. Dicyclohexylcarbodiilllide (G.2 cJ.) dissolved
in dichloromethane (15 ml.) is added followed immediately
by a solution of 3-acetylthio-2-methylpropanoic acid (4.9 g.)
in dichloromethane ~5 ml.). After 15 minu~es s~irriny in
the ice bath and 16 hours at room temperaturc, the precipitate
is filtered of~ and the il-trate is concen~rated to dryness
in vacuo. The residue is dissolved in ethyl acetate and washed
neutral. The organic phase is dried over magnesium sulfate
and concentrated to dryness in vacuo. The residue 1-(3-
acetylthio-2-methylpropanoyl)-L-proline tert-butyl ester is
purified by column chroma-tography (silica gel-cllloroform),
yield 7.9 g.
~xample 29
1 (3-~cet lth:o-2-me~h lpro ano l)-L-l'roline
Y ~ Y ~ Y
Procedure ~
The 1-(3-acetylthio-2-methylpropanoyl)-L-proline
tert-butyl ester of ~xample 28 (7.8 y.) is dissolved ln a
mixture of anisole (55 ml.) and trifluoroacetlc acld (110 ml.).
After one hour storage at room temperature the solvent is
removed in vacuo and the residue is preci~itated several
times from ether-hexane. The residue (6.8 g.) is dissolved
in acetonitxile (40 ml.) and dicyclohexylamine (4.5 ml,) is
added. The crystalline salt lS boiled Witil ~resh acetonitrile
(100 ml~), chilled to room temperature and filtered, yield
3.8 g., m.p. (165) 187-188 . This material is recrystallized
from isopropanol [a~D-67 (C 1.4, EtOH). The crystalline
dicyclohexylamiDe salt is suspended in a mixture of 5% aqueous
potassium bisul~ate and ethyl acetate. The or~anic l~hase ls
washed with water and concentrated to dryncss. The residue is
crystallized ~rom ethyl acetatc-hexane to yield the 1-(3-acetyl-
-30-
6~
HA135b
thlo-2-D-metllylpropanoyl-L-proline,m.p.~3-85~ 162~c,1.7,~tOII).
Procedure B
_
3-Acetylthio-2-methylpropanoic acid (8.1 y~) and
thionyl chloride (7 g.) are Mixed and the sus~ensioll is stirred
for 16 hours at room temperature. The reaction mixture is
concentrated to dryness and distilled in v~cuo (b.p 80 ).
This 3-acetylthio-2-methylpropanoic acid chlorlde (5.4 g.)
and 2N sodium hydroxide (15 ml.) are added to a solution of
L-proline (3.45 g.) in normal sodium hydroxide (30 ml.)
chilled in an ice water bath. ~fter 3 hours s'~irring a~
room temperature, the mixture is extra~ted with e~her, the
aqueous phase is acidified and extracted with e-thyl ace~ate.
Tlle or~anic phase is dried over magnesium sulfate ~nd concentrated
to dryness to obtain 1-(3-acetyl-thio-2-DL-metllyl~ropanoyl-L
proline. ~ ~
Procedure C ;
- Methacryloyl chloride (4.16 g.) is added to a solution
o L-proline (3.45 g.) in a mixture of water (100 ml ) and
sodium blcarbonate (12 g.) chilled in an ice water bath, with
vigorous stirring. When the addition is compLeted, the mixture ; ;
is stirred at room temperature for two hours, and then extracted ~ ~
with ether. The aqueous phase is acidified with N hydrochloric ~ -
acid and extracted with ethyl acetate. The organic phase is
concentrated to dryness in vacuo, the residue is mixed with
thiolacetic acid (3.5 g.~, a few crystals of azobisisobuty-
ronitrile are added a~d the mixture is heated on the steam bath
for two hours. The reaction mix-ture is dissolved in~benzene-
acetic acid ~75:25), and applied to a column of silica gel.
Elution witll the same solverl~ mixturc yields thc 1-~3-acetylthio-
2-DL-methylpropanoyl)-L-urolinc~.
-31-
HA13Sb
ample 30
1-(3-Benzoyl~hio-2-metllylpropanoyl)-L-prolille tert-butyl Ester
By substituting 3-benzoylthio~2-methylpropanoic acid for
the 3-acetylthio-2-methylpropanoic acid in t:lle proced~rc
of Example 23, 1-(3~benzoylthio-2-methylpro~anoyl)-L-proline
tert.butyl ester is obtained.
~xample 31
1-(3-Phenylacetylthio-2-met_ylpropanoyl)-L-ProIine ter~-butyI Ester
By substituting 3-phenylacetylthio-2-methylpropanoic
10 acid for the 3-acetylthio-2-methylpropanoic acid in the
procedure of Example 28, 1-(3-phenylacetylthio-2-methylprouanoyl)-
L-proline tert butyl ester is obtained.
xamplc 32
1-(3-Benzoylt;hio-2-methylpropanoyl)-L-proline
By substituting 1-(3-benzoylthio-2-methylpropanoyl)-
L-proline tert-butyl ester for the 1-(3-acetyltllio)-2- ;~
methylpropanoyl)-l-proline tert-butyl ester in Procedure
of Example 29, 1-(3-benzoylthio-2-metllylpropanoyl)-L-
prollne is obtained.
E~ample 33
(3-phenylacetyltllio-~ e~Vl~5lC~ L 1~ L ~
~y substitutin~ 3-phenylacetylthio-2-metllylpropanoyl)-
L-proline tert-butyl ester for 1-(3-acetylthio-2-me~hylpro~anoyl)-
; L-proline tert-butyl ester ln Procedure A of ~xample 2~,
3-phenylacetylthio-2-methylpropanoyl)-L-proline is obtained.
Example 34
1-(3-MercaPto-2-D-metl~ ~ropanoyl)-L-proline
. . . :
1-(3-Mercapto-2-methylpropanoyl)-L~proline is
obtained by treating the product o~ each o~ Examyles 29, 32 and
30 33 as follows:
,
-32-
HA135b
The thioester (0.85 c~.) is dissolved in 5.5 N
methanolic ammonia and the solution is kept at room temperature
for 2 hours. The solvent is removed ln vacuo and the residue
is dissolved in water, applied to a ion e~chanye column on
the H cycle (Dowex 50, analytical grade) and elu~ed witll
water. ~he ~ractions that yive positlve thiol rea~tion are
pooled and freeze dried. The residue is crystalli~ed from
ethyl acetate-hexane, yield 0.3 g. The 1-(3-mercapto 2~-
methylpropanoyl-L-proline has m.p. 103-104 , [~] 131
(C,2,~tOH).
Example 35
1-(3-~cetyltllio 2--methylpropanoyl)-L-Proline Met}lyl ~s~cr ~,~
1-(3-Acetylthio-2-methylpropanoyl) L-proline is
reacted with an ethereal solution o~ diazome-thane accordinc;
to the procedure described in ~xample 3 to obtain 1-(3-acetyl~-
thio-2-methylpropanoyl)-L-proline methyl ester.
Example 36 ~ '
1-(3-Merca~to-2-methYlpropanoyl) L Proline amide ;~
By substituting 1-(3-acetylthio-2-metllylpropalloyl)-
L-proline methyl ester in the procedure of ~xample 4, 1-(3
mercap-to-2-methylpro~anoyl)-L proline amide is obtaincd.
Example 37
3=Acetylthio-2-Benzylpropanoic ~cid
By substituting 2-benzylacrylic acid for the
methacrylic acid in the procedure of Example 2S, 3 acetyl-
thio-2-benzylpropanoic acid is obtained.
38
3 Acetyltl?io-2 benzylpropanoyl) L-Prolille tcr~-buty:L E:s~er
By substituting 3 acetyl~hio-2-ben~ylpropalloic acid for
the 3-acetylthio-2 methylpropanoic acid in tl~e procedure of
~ 6~ HA135b
Example 22, 1-(3-acetylthio-2-benzylpropanoyl)-L-proline
tert-butyl ester is obtained.
Example 39
1-(3~Acet~lthio-2-benzylpropanoyl)-L-Proline
The product of Example 38 is substituted for the 1-(3-
acetylthio-2-methylpropanoyl-L-proline terl-butyl ester in
the Procedure A of Example 29 to obtain 1-(3-acetylthio-2-
benzylpropanoyl)-L-proline.
Example 40
1-(3-Mercapto-2-benzylpropanoyl) L-Proline
1-(3-Acetylthio-2 benzylpropanoyl)-L-proline is treated
with methanolic ammonia according to the procedure of Example :~
34 to obtain 1-(3-mercapto-2-benzylpropanoyl)-L-proline as an
oil, Rf = 0.47 (silica gel, benzene-acetic acid (75:25).
Example 41
1-(3-Mercapto~2-methylpropanoyl)-L-Hydroxy Proline
By substituting L-hydroxy proline tert-butyl ester in
the procedure of Example 28, treating the product according
to Procedure A of Example 29 and then continuing as in Example
34, 1-~3-acetylthio-2-methylpropanoyl)-L-hydroxyproline
tert-butyl ester, l-(3-acetylthio-2-methylpropanoyl)-L-
hydroxyproline and 1-(3-mercapto-2-methylpropanoyl)-L-
hydroxyproline, respecti~ely, are obtained.
Example 42
1-(3-Mercapto-2-methylpropanoyl)-L-Azetidine-2-Carboxylic Acid
By substituting L-azetidine-2-carboxylic acid tert- ~
butyl ester in the procedure of Example 2~, treating the product ~.
according to the Procedure A of Example 29 and thén continuing as .
-3~- .
` ~'1
in Example 34, 1-(3-acetylthio-2-methylpropanoyl)-L-azetidine-
2-carboxylic acid tert-butyl ester, 1,3-acetylthio-2-methyl-
propanoyl)-L-azetidine-2-carboxylic acid and 1-(3-mercapto-
2-methylpropanoyl-L-azetidine-2-carboxylic acid are obtained.
Example 43
1-(3-Mercapto~2-methylpropdnoly)-L~Pipecolic Acid
By substituting L--pipecolic acid in the procedure of
Example 28, treating the product according to Procedure A of
Example 29 and then continuing as in ~xample 34, 1-(3-acetyl~
thio~2~methylpropanoyl)~L~pipecolic acid tert-butyl ester,
1-(3-acetylthio-2-methylpropanoyl)-L-pipecolic acid and 1-(3-
mercapto~2~methylpropanoyl)-L~pipecolic acid, respectively,
are obtained. -~
Example 44
1-(4-Benzoylthiobutanoyl)-L-Proline
To a solution of L-proline (2.88 g.) in normal sodium
hydroxide (25 ml.) chilled in an ice bath, 2N sodium hydroxide
(12.5 ml.) and 4-chlorobutyryl chloride (3.5 g.) are added.
. ~ ~
The reac-tion mixture is stirred at room temperature for 3.5 ~ ~;
hours and a suspension of thiobenzoic acid (3.75 g.) and ~ ;~
potassium carbonate (2.A g.) in water (25 ml.) is added. After
overnight stirring at room temperature, the reaction mixture is
acidified with concentrated hydrochloric acid and extracted with ~
ethyl acetate. The organic layer is dried over magnesium sul- ~ ;
fate and concentrated to dryness in vacuo. The residue is
chromatographed on a column of silica gel with benzene-acetic
acid (7 1). The fractions containing the desired material are
pooled and concentra-ted to dryness, yield 1.35 g. A small
aliquot of this material is dissolved in ethyl acetate and
dicyclohexylamine is added until pH 8-10 (on a wet pH paper).
': ~
~35- ~-
The dicyclohexylamine salt crystallizes out, immediately, m.p.
159-161.
Example ~5
1-(4-Mercaptobutanoyl)-L-Proline
1-(4-Benzylthiobutanoyl)-L-proline (1 03 g.) is
dissolved in a mixture o water (4 ml.) and concentrated
ammonia (2.7 ml.). A~ter one hour stirring at room tempera-
ture, the mixture is diluted with water, filtered, extracted with
ethyl acetate, and the aqueous phase was concentrated in vacuo.
This ammonium sal-t of 1-(4-mercaptobutanoyl)-L--proline is
purified by ion e~change chromatography on a column of diethyl-
aminoethyl-Sephadex (cross lin~ed dextran) with a gradient of
ammonium bicarbonate, yield 0.7 ~. The ammonium salt is
dissolved in water (2 ml.) and applied to a column of
Dowex 50 sulfonic acid resin analytical grade in the hydrogen
form, and the free acid is eluted with water. The fractions
containing the desired material (sulfhydryl reagent and
carboxyl reagent positive) are pooled and freeze dried to
obtain l-(4-mercaptobutanoyl)-L-proline. The dicyclohexyl
ammonium salt is produced by the procedure of Example 44,
m.p. 157-158.
Example 46
- ~ :
4-Bromo-2-Methyl~utanoic Acid
Ethyl-4-bromo-2-methylbutanoate [G. Jones and J. Wood,
Tetrahedron, 21, 2961 (1965)] (1.04 g.) is dissolved in
dichloromethane (50 ml.) and cooled to -10. A 1 M solution
or boron tribromide in dichloromethane (50 ml.) is added
dropwise with stirring and the stirring is continued for
1 hour at -10 and 2 hours at 25. The reaction is terminated
-36-
- l~O~B64
by the careful addition of water. The layers are separated
and the organic phase is washed with water, dried and concen-
trated to dryness to obtain 4-bromo-2-methylbutanoic acid.
Example 47
1-(4-Benzoylthio-2-methy~lbutanoyl)-L-Proline
a) 4-Bromo-2-methylbutanoic acid (8 g.) and thionyl chloride
(7 g.) are mixed and the mixture is stirred Eor 16 hours at room
temperature. The reaction mixture is concentrated to dryness
and distilled in vacuo.
.
b) To a solution of L-proline (2.88 g.) in normal sodium
hydroxide (25 ml.) chilled in an ice bath, 2N sodium hydroxide
(12.5 ml.) and the 4-bromo-2-methylbutanoic acid chloride
obtained in part (a) (3.9 g.) are added. The reaction - -
mixture is stirred at room temperature for 3.5 hours and a
suspension of thiobenzoic acid (3.75 g.) and potassium
carbonate (2.4 g.) in water (25 ml.) is added. After overnight
stirring at room temperature, the reaction mixture is acidified
with concentrated hydrochloric acid and extracted with ethyl
acetate. The organic layer is dried over magnesium sulfate
and concentrated to dryness in vacuo. The residue is
chromatographed on a column of silica gel with benzene-acetic
acid (7:13. The fractions containing the desired product,
l-(4-benzoylthio-2-methylbutanoyl)-L-proline are pooled
and concentrated to dryness in vacuo.
Example 48
1-(4-Mercapto-2-methylbutanoyl)-L-Proline ~-
By substituting 1-(4-benzoylthio-2-methylbutanoyl)-
L-proline for the 1-(4-benzoylthiobutanoyl)-L-proline-in
the procedure of Example 45, l-(mercapto-2-methylbutanoyl)-
L-proline in obtained.
-37-
~J ~
'`
ExampLe 49
4-Bromo 2-benzylbutanoic acid
By substituting ethyl-4-bromo-2-benzylbutanoate
~prepared by thc procedure of G. Jones and J. Wood [Tetrahedron,
21, 2961 (1965) starting with diethylbenzylmalonate]] for
the ethyl-4-bromo-2-methylbutanoate in the procedure of
Example 46, 4-bromo-2-benzylbutanoic acid is obtained.
Example 50
l-(4-Benzoylthio-2-benzylbu-tanoyl) L-Proline
By substituting 4-bromo-2-benzylbutanoic acid for
the 4-bromo-2-methylbutanoic acid in the procedure of
Example 47, 1-(4-benzoylthio-2-benzylbutanoyl)-L-proline
is obtained.
Examp~e 51
1-(4-Mercapto-2-benzylbutanoyl)--L-Proline
By substituting 1-(4-benzoylthio-2-benzylbutanoyl)-L-
proline for the l-(4-benzoylthiobutanoyl)-L-proline in the
procedure of Example 45, 1-(mercapto-2-benzylbutanoyl)- -
L-proline is obtained.
Example 52
1-(4-Mercaptobutanoyl)-L-Hydroxyproline
By substituting L-hydroxyproline for -the L-proline
in the procedure of Example 44 and subjecting the product to
ammonolysis as in Example 45, l-(4-benzoylthiobutanoyl)-L-
hydroxy-proline and l-(4-mercaptobutanoyl~-L-hydroxyproline,
respectively, are obtained.
_ample 53
l-(4-Mercaptobutanoyl)-L-Azetidine-2-Carboxylic Acid --
By substituting L-azetidine-2-carboxylic acid for the
L-proline in the procedure of Example 44 and subjecting the
-38-
l~ `
,
364 ~ ~
product to ammonolysis as in Example 45, l-(4-benzoylthio-
butanoyl)-L-azetidine-2-carboxylic acid and l-(~-mercapto-
butanoyl)-L-azetidine-2-carboxylic acid, respectively, are
obtained~
E~
1=(4-Mercaptobutanoyl)-L-Pipecolic Acid
By substituting L-pipecolic acid for the L-proline in
the procedure of Example 44 and subjecting the product to
ammonolysis as in Example 45, 1-(4-benzoylthiobutanoyl)-L-
pipecolic acid and 1-(4-mercaptobutanoyl)-L-pipecolic acid,
:
respectively, are obtained.
~., . ~ .
Example 55 ~;
1-(3-Acetylthiobutanoyl)-L-Proline tert-butyl Ester
Dicyclohexylcarbodiimide (6.2 g.) and 3-acetylthio-
butyric acid (4.86 g.) are added to a solution of L-proline
tert-butyl ester (5.1 ~.) in dichloromethane (60 ml.) stirred
in an ice bath. After 15 minutes the ice bath is removed
, ,
and the mixture is stlrred at room temperature for 16 hours. ~;
The precipitate is filtered, the filtrate is concentrated to
dryness and the residue is chromatographed on a column of `~
silica gel with chloroform to obtain l-(3~acetylthiobutanoyl)-
L-proline tert-butyl ester, yield 5.2 g. ~;
Example 56 ;
1-(3-Acetylthiobutanoyl)-L-Proline
The l-(3-acetylthiobutanoyl)-L-proline tert-butyl ester ~
of Example 55 (5.2 g.) is dissolved in a mixture of trifluoro- ~.
acetic acid (60 ml.) and anisolé (30 ml.) and the solution
is kept at room temperature for one hour. The solvents are removed
in vacuo and the residual l-(3-acetylthiobutanoyl)-L-prollne
is reprecipitated from ether-hexane several times, yield - -~
~`
-39-
4 g.. The dicyclohexylamine salt is made by the procedure
of Example 44, m.p. 175-17~.
Example 57
1-(3-Merc ptobutanoyl)-L-Proline
The 1-(3-acetylthiobutanoyl)-L-proline of
Example 56 (0.86 g.) is dissolved in 5~5 N. methanolic
ammonia (20 ml.) and the reaction mixture is stored at room
temperature for 2 hours. The solvent is removed in vacuo
and the residue chromatographed on an ion exchange column
(Dowex 50) with water. The fractions containing the desired
1-(3-mercaptobutanoyl)-L-proline are pooled and lyophilized,
yield 0.6 g. The dicyclohexylamine salt is produced by the
procedure of Example 44, m.p. 183-184.
Example 58
1-[3-[[(Ethoxy)carbonyl]thio]propanoyl]-L-proline
Ethyl chloroformate ~1.2 g.) is added to a solution of
3~mercaptopropanoyl-L-proline (2.03 g.) in normal sodium bicar-
bonate (30 ml.) and the mixture is stirred vigorously at 5
for one hour, and for two hours at room temperature. After
acidification with concentrated hydrochloric acid, the mixture
is extracted with ethyl acetate. The organic phase is
washed with water, dried over magnesium sulfate, and concentra-
ted to dryness to yield 1-[3-[[(ethoxy)carbonyl~thio]propanoyl]-
L-proline.
-40-
'
~'
~ :
Example 59
1-[3-[[(E-thoxy)thiocarbonyl]-thio]propanoyl]-L-proline
Aqueous 2N sodium hydroxide (25 ml) and 3-hromopropionyl
chloride (8.5 g) are added to a solution of L-proline (5.75 g)
in N sodium hydroxide (50 ml) chilled and s-tlrred in an ice
bath. After five minutes -the ice bath i5 removed and the
stirring is continued at room temperature. After three hours
ethyl xantogenic acid potassium salt ~9.6 g) is added and the
mixture is stirred overnigh-t at room temperature. The solution ~ ~ -
is acidified with concentrated hydrochloric acid and extracted
with ethyl acetate. The organic layer is concentrated to dry-
ness and the residue is chromatographed on a column o~ silica
gel with a mixture of benzene-acetic acid (7:1) as solven-t, to ~;
yield 1-[3-[[(ethoxy)thiocarbonyl]thio]propanoyl]-L-proline,
p. 94_95o.
Example 6~
1-[3-[[(Benzylthio)carbonyl]thio]propanoyl]-L-proline
A solution o~ benzylthiocarbonyl chloride (11 ml) in
dioxane (20 ml) is added in five portions to a solution of
1-(3-mercaptopropanoyl)-~-proline (1.6 g) in normal sodium
bicarbonate (24 ml) chilled in an ice bath, over a period of
30 minutes. The ice bath is removed and the stirring is
continued for 2.5 hours at room temperature. After acid-
ification with concentrated hydrochloric acid, the aqueous
phase is extrac~ed with ethyl acetate. The organic phase
is dried over magnesium sulfate and concentrated to dryness
to yield 1-[3- E [ (benzylthio)carbonyl]thio]propanoyl]-~-proline.
-41-
.
.~ . .
_ample 61
1-[3-[[(Ethylthio)thiocarbonyl]thio]propanoyl~-
.
Aqueous 2N sodium hydroxide (25 ml) and 3-bromopropionyl
chloride (8.5 g) are added to a solution of L-proline (5.75 g)
in N sodium hydroxide (50 ml~ chilled and stirred in an ice
bath. After five minutes, the ice bath is removed and the
stirring is continued at room temperature. After three hours,
ethyl trithiocarbonate potassium salt (10.5 g~ is added and
the mixture is stirred at room temperature overnight. After
acidification with concentrated hydrochloric acid, the mixture
is extracted with ethyl acetate. The organic layer is dried
over magnesium sulfate and concentrated to dryness to yield
1-[3-[[(ethylthio]t~iccarbonyl]thio]propanoyl]-L-proline.
Example 62 ;
3~[[(Methylamino)thiocarbonyl]thio]propionic acid
~ ethylisothiocyanate (4 g) is added to a solution of
3-mercaptopropionic acid (5.3 g) in a mixture of pyridine
(250 ml) and 0.5 N sodium hydroxide (100 ml). The solution
is kept at 40 for two hours and concentrated to dryness
in vacuo. The residue is dissolved in water (100 ml.),
_
acidi-fied with concentrated hydrochloric acid and extracted
with ether. The organic phase is concentrated to dryness
to yield 3-[[(methylamino)thiocarbonyl]thio]propionic acid,
m.p. 86-87.
-42-
Example_ 3
1~[3-[[(Methylamino)thiocarbonyl]thio~propanoyl]-L-prollne
tert-butyl ester
To a solution of L-proline tert-butyl ester ~1.71 g) and
hydroxybenzotriazole (1.35 g) in dichloromethane (lO m]) chilled
and stirred in an ice bath, dicyclohexylcarbodiimide (2.06 gl
and 3-methylaminothiocarbonylthiopropionic acid (1.79 g) are
added. A~ter 15 minutes, the bath is removed and the stirring -
is continued overnight. The precipitate is filtered off and
the filtrate is diluted with ethyl acetate and washed neutral.
The organic phase is concentrated to dryness to yield
l-[3-[[(methylamino)thiocarbonyl]thio]propanoyl]-L-proline
tert-butyl ester, m.p. 129-130.
Example 64
l-[3-[[(Methylamino)thiocarbonyl]thio]propanoyl]-L-proline
A) l-(MethylaminothiocarbonylthiopropanoyI)-L-proline tert- `
butyl ester (0.98 g) is dissolved in a mixture of anisole (3.6 ~ ;
ml) and trifluoroacetic acid (7.5 ml). After one hour at room
temperature the mixture is concentrated to dryness in vacuo and
the residue precipitated from ether-hexane three times. This
material is chromatographed on a column of silica gel with a
solvent mixture of benzene~acetic acid (75:25) to yield 1~[3-
[[(methylamino)thiocarbonyl]thio]propanoyl]-L-proline, ;
Rf = 0.4 [silica gel-benzene:acetic acid (75:25)]. The
dicyclohexylammonium salt has m.p. 127-129.
~ .
-43-
;~
.: .
Gg~
~) Methylisothiocyanate (4 g) ls added to a solution of 3-
mercaptopropanoyl-L-proline (10.1 CJ) in a mixture of pyridine
(250 ml) and 0.5 ~ sodium hydroxide (lO0 ml). The solution is
kept at 40 for two hours and concentrated to dryness ln vacuo.
The residue is dissolved in wa-ter (100 ml), acidified with con-
centrated hydrochloric acid and extra~ted with ethyl acetate.
The organic phase is concentrated to dryness to yield
l-~3-[[(methylamino)thiocarbonyl]thio]propanoyl]-L-proline.
Example 65
l-_[3-[[(Ethylamino)carbonyl]thio]propanoyl]-L-proline
Ethylisocyanate (0.45 ml) is added to a solution of
l-~3-mercaptopropanoyl)-L-proline (1 g) in a mixture of N
sodium hydroxide t5 ml) and pyridine (5 ml). The solution
is heated at 40 for four hours and concentrated ln vacuo.
The residue is distributed between 0.1 N hydrochloric acid
and ethyl acetate. The organic layer is washed with water,
dried over magnesium sulfate and concentrated to dryness to
yield 1-[3-[[(ethylamino)carbonyl]thio]propanoyl]-L-proline.
The dicyclohexylammonium salt is prepared by adding dicyclo-
hexylamine to a solution of the free acid in ethyl acetate, ~;~
m.p. 150-152.
Examp_e_66 '
1_[3-[[(Ethoxy?carbonyl]thio]-2-methylpropanoyl]-L-proline ~ `
By substituting 1-(3-mercapto-2-methylpropanoyl]-L-proline
for the 3-mercaptopropanoyI-L-proline in the procedure of
Example 58, l-[3-[~(ethoxy)carbonyl]thio]-2-methylpropanoyl]-L-
proline is obtained~
-44-
~, ~
flA135b
arnpl.e 67
1-[3-[[~thoxy)carborlyl]thio]butanoyl]-1.-proline
By substituting 1-[3-mercaptobutanoyl)-L-proline for the
3-mercaptopropanoyl-L-proline in the procedure of ~,cample 58, 1-
3-[[(ethoxy)carbonyl]-thlo~butanoyl]-L-proline is obtained.
Examplc 68
1-[3-[[(Ethoxy)thiocarbonyl]thioJpropanoyl]-L-azetidine-2-
carboxylic acld
By substituting L-azetidine-2-carboxylic acid for
L-proline in the procedure o~ Example 59, 1-[3-[[(ethoxy)-
thiocarbonyl]thioJpropanoyl]-L-azetidine-2-carboxylic acid
is obtained.
E'x rnple 69
1-[3-[[(~thoxy)thiocarbonyl]thio]propanoyl]-L-pipecolic acid ~ -
By substituting L-pipecolic acid for L-proline in the
procedure of Example 59, 1-[3-[[(ethoxy~thiocarbonyl]thio]pro-
panoyl]-L-pipecolic acid i5 obtained.
Example 70
1-[4-[~(Benzyltllio)carbonyl]tllio]butalloy1J-I,-pLo]illc
. .
,
By substitutin~ 4-mercaptobutanoyl-L-proline ~or the 3-
mercaptopropalloyl-L-proline in the proc-edure of ~xaml)lc 60, I-
[4-[[(benzylthio]carbonyl]thio]bu~anoyl]-L-proline is obtained.
~xample 71
1-[2-[[(~enzylthio)carbonyl]thlo]propanoyl]-L-!jroline
By substitutin~ 2-mercaptopropanoyl-L-prolirle for the 3-
mercaptopropanoyl-L-proline in the procedurc of ~xa~ple 60, 1-
[2~[[(benzyltllio)carbonyl~thio]propanoyl]-L-prolille is ob~ained.
-45-
164
Examp_e 72
1-[3-[[(E-thylthio)thiocarbonyl]thio]propanoyl]-L-proline methyl
ester
A solution of 1-[3-[[(ethyl-thio)thiocarbonyl]thio~-
propanoyl]-L-proline in ethyl acetate i5 treated with an ethereal
solution of diazomethane until persistent yellow color. After
discharginy the yellow color with a few drops of acetic acid, ;
the solvents are removed in vacuo to yield l--[3-[[(ethylthio)-
thiocarbonyllthio]propanoyl]-L-proline methyl ester.
Example 73
1-[3-[[(methylamino)thiocarbonyl]thio]propanoyl]-5-hydroxy-L-
pipecolic acid
By substituting 1-(3-mercaptopropanoyl)-5-hydroxy-L-
pipecolic acid for the 3-mercaptopropanoyl-L-proline in the
Procedure B of Example 64, 1- E 3-[[(methylamino)thiocarbonyl]thio]-
propanoyl]-5-hydroxy-L-pipecolic acid is obtained.
Example 74
1-[3-[ E (Methylamiho)thiocarbonyl]thio]-2-methylpropanoyl]-L-
proline amide
By substituting 1-(3-mercapto-2-methylpropanoyl)-L-
proline amide for the 3-mercaptopropanoyl-L-proline in the
Procedure B of Example 64, 1-[3-[[(methylamino)thiocarbonyl]thio]-
2-methyl-propanoyl]-L-proline amide is obtained.
Example 75
1-[3-[[(Phenoxy)carbonyl]thio]propanoyl]-L-proline
By substituting phenylchloroformate for ethyl chloroformate
in the procedure of ~xample 5~, 1-[3-[[(phenoxy)carbonyl]thio]-
propanoyl]-I.-proline is obtained. -
-46-
Example 76
1-[3 [~(Phenoxy)carbonyl}thio]butanoyl]-L-proline
By substituting phenylchloroformate for the ethyl
chloroformate and 4-mercaptobutanoyl-L-prolirle for the
3-mercaptopropanoyl-L-proline in the procedure of Example 58,
1-[3-[[(phenoxy)carbonyl]thio]butanoyl]-~-proline is obtained.
Example 77
1=[3-[[(Phenylamino)carbonyl]thio]propanoyl]-L-proline
By substituting phenylisocyanate for the ethylisocyanate
in the procedure of Example 65, 1-[3-[~(phenylamino)carbonyl]-
thio]propanoyl] L-proline is obtained.
Example 78
1-[3-[[(Phenethylamino)carbonyl]thio]propanoyl]-L-proline
:
By substituting phenethylisocyanate for the ethylisocyana-te
in the procedure of Example 65, 1-[3-[[(phenethylamino)carbonyl]-
thio]propanoyl]-L-proline is obtained. -~
Example 79
.
1 [3-[[(Ethylamino)carbonyl]thio]-2-benzylpropanoyl]-L-proline
By substituting 1-(3-mercapto-2-benzylpropanoyl)-L-
proline for the l-(3-mercaptopropanoyl)-L-proline in the
procedure of Example 65, 1-[3-[[(ethylamino)carbonyl]thio]-
2-benzylpropanoyl~-L-proline is ohtained.
., ~
-47-
64
Example 80
l-(3-Methyl-thiopropanoyl)-L-proline
A) Methyl 3-methyl-thiopropionate (51 g) is saponiEied with
a 10% sodium hydroxide solution (150 ml, 30 minutes at 100).
The cooled solu-tion is extracted with ether and then acidified.
The crude acid thus obtained is distilled and converted to
the acid chloride with thionyl chloride.
A solution of L-proline (11.5 g) in N sodium hydroxide ~100 cc)
is chilled in an ice bath and the 3-methylthiopropanoic acid
chloride (6.9 g) is added dropwise with viyorous stirring over
a ten minutes period. ~fter five hours the reaction mixture
is acidified and extracted with ethyl ether to yield l-(3-
me-thylthiopropanoyl)-L-proline. The dicyclohexylammonium salt
is prepared by adding dicyclohexylamine to a solution o~ the
free acid in e-thyl acetate, m.p. 169-171.
B) Methyl iodide (71 g) is added to a solution of 1-~3-
mercaptopropanoyl)-L-proline ethyl ester (115 ~) and sodium
(11.5 g) in ethanol (400 ml). The reaction is allowed to
proceed overnight, the ethanol is removed in vacuo and the
residue is dissolved in a mixture of ethyl acetate and water.
The organic layer is dried and concentrated to dryness ln vacuo.
The resulting 1-~3-methylthiopropanoyl)-L-proline ethyl ester
(98 g) is suspended in a mixture o~ methanol (200 ml) and 5 N
sodium hydroxide (200 ml) and stirred at room temperature for
five hours. The methanol is removed ln vacuo, and the aqueous
phase is extracted with ethyl acetate, acidified and ~eextracted
with ethyl acetate. This last organic phase is washed with
water, dried and concentrated to dryness to yield l-(3-methylthio-
propanoyl)-L-proline.
-48-
.
.
Example 81
~=~ :
Aqueous 2 N sodium hydroxide (25 ml) and 3-bromopropionyl
chloride (8.5 g) are added to a solution of l-proline (5.75 g)
in N-sodium hydroxide (50 ml) chilled and stirred in an ice
bath. After five minutes~ the ice bath is removed and the
stirring is continued for three hours at room temperature. The
reaction mixture is acidified with concentrated hydrochloric acid
~extracted with ethyl acetate. The organic layer is washed
with water, dried and concentrated to dryness ln vacuo. The
residue is dissolved in a mixture of 4-chlorobenzenethiol (8 g),
sodium hydroxide (4.2 g) and ethanol (300 ml). The solution is
refluxed for 6 hours. The solvent is removed ln vacuo and the
residue is dissolved in water, acidified with concentrated hydro-
chloric acid and extracted with ethyl acetate. The or~anic layer
is washed with water, dried, and concentrated to dryness ln
vacuo to yield 1-[3-~4-chlorophenylthio)propanoyl]-L-proline.
Example 82
1,[[(3-Benzylthiomethyl)thio~propanoyl]-L-proline
1-(3-Mercaptopropanoyl)-L-proline (8.1 g) is dissolved
in boiling liquid ammonia (100 ml) and small pieces of sodium
are added until permanent blue color is obtained which is then
discharged with a small piece of ammonium chloride. Benzyl-
thiomethyl chloride (6.9 g) is added and the ammonia is allowed
to evaporate. The final traces of ammonia are removed ln
vacuo, the residue is dissolved in water and extracted with
ethyl acetate. The aqueous phase is acidified with concentrated
hydrochloric acid and extracted with ethyl acetate. The organic
layer is washed with water, dried and concentrated to dryness
-49-
~ .
~ .
to yield l-[[(3-benzylthiomethyl)thio]pxopanoyl]-L-proline.
Example 83
1-[[(3-Acetamidomethyl)thio]propanoyl]-L-proline
1-(3-Mercaptopropanoyl)-L-proline (2 gl and N-hydroxy-
methylacetamide (0.89 g) are dissolved in trifluoroacetic acid
(10 ml) and the solution is stored at room temperature for one
hour. The excess tirfluoroacetic acid is removed ln vacuo and
the residue is precipitated several times from ether-hexane.
Finally, the residue is distributed between dilute hydrochloric
acid and ethyl aceta-te~ The organic layer is washed with water,
dried and concentrated to dryness to yield l-[[3-acetamido-
methyl)thio]propanoyl]-L-proline.
Example 84
.
l-(Methylthioacetyl)-L-proline
S
By substituting methyl mè~thylthioacetate for the methyl
3-methylthiopropionate in the Procedure A of Example 80, 1-
(methyIthioacetyl)~L-proline, m.p. 123-124, is obtained.
~xample 85
l-(Benzylthioacetyl)-L-proline
By substituting benzylthioacetyl chloride for the 3-
methylthiopropanoyl chloride in the Procedure A of Example 80,
l-(benzylthioacetyl)-L-proline, m.p. 86-88, is obtainea.
-50-
3L~ 4
HA135b
E~ample 86
-
1-~3-[(2-~henylc~hyl)t}lio]~ropanoyl]-rl-pro~ e
_
By substitutincJ phenethylbromide for thc metllyl iodidc in
the Procedure B of Example 80, 1-[3-~(2-pllellyletllyl)~hio)proparloyl]-
L-proline is obtained.
.
~xample 87
1-[3-[(Triphenylmcth l)~hio~pro~anoyl]-L-prolinc
By substituting triphenylmethyl chloride ~or thc metllyl
iodide in the Procedure B of Example 80, 1-[3-[(tri~henylmethyl)-
thio]propanoyll-L-proline is obtained.
Example 88
___
1-(3-~lethylthio-2-lnetllylpropanoyl)-L,-prolirle amiclc
By substitutiny 1-(3-mercapto-2-methylpropanoyl)-L-proline
amide for the 1-(3-mercaptopropanoyIj-L-proline ethyl estcr in
the Procedure B o~ Example 80 and eliminatillc~ the saponification
step, I-(3-methylthio-2-methylpropanoyl)-L-r?roline amide is
obtained.
~xample 89
1- (3-~;ethylthiol?ropalloyl)-I,-aze~:icl~ e-2-carboxy:Lic ac,icl :
By substi-tuting L-azetidine-2-carboxylic acid for the L-
proline in the Procedure A of Example 80,1-(3-rr,ethylthioproPanoyl)-
L-azetidine-2-carboxylic acid is obtained. ~,
.
'.
-51-
... . : .
', :
i4
Example 90
1-[3-(4-Methoxyphenylthio)propanoyl~-L-proline
By substituting 4-methoxybenzenethiol for the 4-chloro-
benzenethiol in the procedure of E~ample 81, 1~[3-(4-methoxy-
phenylthio)propanoyl] L-proline is obtained.
Example 91
l-(3-Methylthiopropanoyl)-L-pipecolic acid ~
,,
By substituting L-pipecolic acid for the I.-proline in
the Procedure A of Example 80, 1-(3-methylthiopropanoyl)-L- ~;
pipecolic acid is obtained.
Example 92
1-[2-(4-Chlorophenylthio propanoyl]-L-proline
.
By substituting 2-bromopropionyl chloride for the
3-bromopropionyl chloride in the procedure of Example 81,
1-[2-(4-chlorophenylthio-propanoyl~-L-proline is obtained.
Example 93
1-[3-[(Diphenylmethyl)thio]-2-benzylpropanoyl)]-L-proline
Diphenylmethanol (0.92 g) and 1-(3-mercapto-2~benzyl-
propanoyl)-L-proline (1.5 g) are dissolved in trifluoroacetic
acid (10 ml) and the solution is kept at room temperature for
30 minutes. The excess trifluoroacetic acid is removed ln vacuo
to yield l-[3-[(diphenylmethyl)thio]-2-benzylpropanoyl]-L-proline.
. .
-52-
~ ~ .
. ,, , ;, ~
~1.9.'f~ 364
Example 94
1-~4-(4-Chlorophenylthio)butanoyl]-L-proline
.
By substitutlng 4-bromopropionyl chloride for the 3-
bromopropionyl chloride in -the procedure of Example 81, 1-[4-(4-
chlorophenyl-thio)butanoyl]-L-proline is ob-tained.
Example 95
1-[3-[(Benzylthiomethyl)thio]butanoyl]-L-proline
By substituting 3-mercaptobutanoyl-L-proline for the
3-mercaptopropanoyl-L-proline in the procedure of Example 82,
1-[3-[(benzyl-thiomethyl)thio]butanoyl]-L-proline is obtained.
Example 96
1-[[4-[(Acetamidome hyl)thio]-2-methylbutanoyl~-L-proline
By substituti.ng 1-(4-mercapto-2-methylbutanoyl)- :~
L-proline for the 1-(3-mercaptopropanoyl)-L-proline in the
procedure of Example 83, 1-[[4-(acetamidomethyl)thio]-2-
methylbutanoyl]-L-proline is obtained.
Example 97
1-[3-(Ethyldithio)propanoyl]-L-proline
:
A) 3~Mercaptopropanoyl-L-proline (10 g) is added to a
solution of ethylthiosulfinate (8.4 g) in methanol (100 ml)
and the reaction mixture is stirred vigorously at room ..
temperature for four hours. The methanol is re.moved in
vacuo to yield l-(3-ethyldithiopropanoyl)-L-proline.
'~
B) A solution of ethylthiosulfinate (8.4 g) in : .
ethanol (50 ml) is added to an aqueous solution of 3-mercapto-
propanoyl-L-proline (10 g) maintained at pH 6-7 by careful
: .
addition of sodium hydroxide. The mixture is stirred vigorously ~:
-53-
.
- ' : .' :
at room temperature until negative thiol reaction. The mix-
ture is diluted with water, adjusted to pH 8 and extracted
with ethyl acetate, the aqueous phase i5 acidified to pH 3
and extracted again with ethyl acetate. This lattex extract
is washed with water, dried and concentrated to dryness -to
yield 1-~3-(ethyldithio)propanoyl]-L-proline.,
Example 98
1-[3-[(4-Methylphenyl)dithio]propanoyl]-L-pro'llne
A solution of 4-methylphenylsulfenyl chloride (1.76 g.) ~'~
in ether (20 ml) is added to a solution of 3-mercaptopropanoyl-
I.-proline (2 g) in 0.5 N sodium hydroxide (20 ml) chilled in
an ice bath. The mixture is stirred vigorously for one hour,
and the aqueous phase is separated, acidified with concentrated
hydrochloric acid and extracted with ethyl acetate. The organic
phase is washed with water, dried and concentrated to dryness
to yield 1-[[3-(4-methylphenyl)dithio]propanoyl]-L-Proline.
Example 99
1-[3-~Phenyldithi'o-propanoyl}-L-proline
By substituting phenylthiosulfinate [prepared from phenyl-
disulfide according to U. Weber and P. Eartter, Z. Physiol. Chem.,351, 1384 (1970)] for the ethylthiosulfinate in the procedure of
Example 97, 1-[3-(phenyldithio-propanoyl]-L-proline is obtained.
Example 100 ~ '
1-~3-[(2-Phenylethyl~dithio]propanoyl]-L-proIine
By substituting 2-phenylethylthiosulfinate (prepared from
phenethyldisulfide) for the ethylthiosuflinate in the procedure ~,
of Example g7, 1-[3-[(2-phenylethyl)dithio]propanoyl]-L-proline
is obtained.
-54-
~ . . .
.
6~
Example 101
.
l-[3-[(2-Hydroxye-thyl)dlthio]propanoyl]-L-proline
To a solution of 1,1'-[(sulfinylthio)-bis-(3-propanoyl)]-
bis-L-proline (21 g) in methanol (100 ml), mercap-toethanol(4.2 g)
is added and the reaction mixture is stirred vigorously at room
temperature for four hours. The me-thanol is removed in vacuo
and -the residue is pruified by chromatography on a silica gel
column to yield 1-[3-[(2-hydroxyethyl)dithio]propanoyl]-L-proline.
Example 102
1-[2-(Ethyldithio)propanoyl]-L-proline
` ~ :
By substituting 2-mercaptopropanoyl-L-proline for 3-
mercaptopropanoyl-L-proline in the procedure oE Example 97,
1-[2-(ethyldithio)propanoyl]-L-proline is obtained.
Example 103
1-[3-[(~-Methylphenyl)dithio~utanoyl]-I-proline `~
By substituting 3-mercaptobutanoyl-L-proline for the 3-
mercaptopropanoyl-~-proline in the procedure of Example 98, ~ ~
l-[3-(4-methylphenyl)dithio]butanoyl]-L proline is obtained. -
Exampl`e 104
l-[3-(Ethyldithio)-2-methylpropanoyl]-I,-proline methyl ester
.
By substituting l-(3-mercapto-2-methylpropanoyl)-L-proline
for the 3-mercaptopropanoyl-L-proline in the procedure of Example
97 and then treating the product with ethereal diazomethane as
in the procedure of Example 72, 1-[3-(ethyldithio)-2-~ethylpro-
panoyl]-L-proline methyl ester is obtained.
-55-
~ ':
.
Example 105
l-[3-(Ethyldithio)propanoyl]-L-azetidine-2-carboxylic acid
By suhstituting 3-mercaptopropanoyl-L-azetidine-2-
carboxylic acid Eor the 3-mercaptopropanoyl-L-proline in the
procedure of Example 97, 1~[3-(ethyldithio)propanoyl]-L-aze-
tidine-2-carboxylic acid is obtained.
Example 106
1-[3-[(4-Methylphenyl)dithio]-2-methylpropanoyl]-L-hydroxyproline
:
By substituting 1-(3-!mercapto-2-methylpropanoyl)-L-
hydroxy proline for the 3-mercaptop~opanoyl-L-proline in the
procedure of Example 98, 1-~(3-[4-methylphenyl)dithio]-2-
methylpropanoyl]-L-hydroxyproline is obtained.
Example 107
l-~4-~Ethyldithio)butanoyl]-L-pipecolic acid
By substituting 4-mercaptobutanoyl-L-pipecolic acid for
the 3-mercaptopropanoyl-L-proline in the procedure of Example 97,
l-~4-(ethyldithio-butanoyl]-L-pipecolic acid is o~tained.
Example 108
1-[3-(Ethyldithio)propanoyl]-5-hydroxy-L-pipecolic acid
By substituting 1-(3-mercaptopropanoyl)-5-hydroxy-L-
pipecolic acid for the 3-mercaptopropanoyl-L-proline in the
procedure of Example 97, 1-[3-(ethyldithio)propanoyl]-5-hydroxy-
L-pipecolic acid is obtained.
,
-56-
~,.-~ .
Example 109
1-[3-[(2-Amino-2-carboxyethyl)di hio]propanoyl]-L-proline
A 0.5 M solution of thiocyanogen in glacial acetic acid
is prepared by shaking for ten minutes in a sealed flask 600 mg
of dry lead thiocyanate with a solution o 75 ~1 of bromine in
3 ml of acetic acid. After removal of lead bromide and excess
lead thiocyanate by centrifugation, 2.5 ml of this solution is
mixed with 2.5 ml of a 0.41 M solution of cysteine hydrochloride ~i
previously neutralized with dilute sodium hydroxide. This
mixture is immediately added to 0.75 ml of a 1.9 M solution of
3-mercaptopropanoyl L-proline previously neutralized with dilute i
sodium hydroxide. After twenty minutes the mixture is titrated
to incipient brown color with alcoholic iodine, and adjusted to
pH 3. The precipitate is removed by fiItration and the filtrate
is applied to a column of cation exchange resin (Dowex 50~. The
column is washed with water until no more acidic material is ~;~
removed and then eluted with pyridine-acetate buffer pH 6Ø ~ '
The fractions containing the disulfide of cysteine and 3-
mercaptopropanoyl-L-proline are pooled and concentrated to
dryness.
Example 110
1,1'-~Dithiobis)4~propanoyl)]-bis-L-proline
3-Mercaptopropanoyl L-proline (0.95 g) is dissolved in
water (20 ml) and the pH is adjusted to 6.5 with N-sodium
hydroxide. An ethanolic solution of iodine is added drop- ~;
wise while maintaining the pH at 6.5 with careful addition
of N sodium hydroxide. When a permanent yellow color_is
obtained the addition of iodine is stopped and the color
-57-
, .
is discharged with a small amount of sodium thiosulfate.
The reaction mixture is acidified with concentrated h~dro-
chloric acid and extracted with ethyl aceta-te. ~he organic
phase is washed with water, dried and concentra-ted to dryness
to yield l,l'-[dithiobis(3-propanoyl)]-bis-L--proline. The di-
cyclohexylammonium salt is prepared by additlon o~ dicyclo-
hexylamine to a solution of the free acid in acetonitrlle,
m.p. 179-180.
Example 111
1,1'-[Dithiobis(2-D-methyl-3-propanoyl)]-bis-L-proline
By substituting 3-mercapto-2-D-methylpropanoyl-L-
proline for the 3-mercaptopropanoyl~L-proline in the procedure
of Example llO~ 1,1'-[dithiobis~2-D-methyl-3-propanoyl)]-bis-
L-proline is obtained, m.p. 236-237.
Examp~l'e 112
'l,l'-[Dithiobis(2-propanoyl)-bis-L-proline
By substituting 2-mercaptopropanoyl-L-proline for the
3-mercaptopropanoyl-L-proline in the procedure of Example 110,
1,1'-[dithiobis)2Npropanoyl)]-bis-L-proline is obtained.
Example 113
l,l'-(Dithiobisacetyl)-bis-L-hydroxy proline
By substituting 1-(2-mercaptoacetyl)-L-hydroxyproline
for the 3-mercaptopropionyl-L-proline in the procedure of ~ -
Example llO, l,l'-(dithiobisacetyl)-bis-L-hydroxyproline is '
obtained.
Example 114 ,~
1,1'-(Dithiobisacetyl)-bis-L-azetidine-2-carboxylic ac~id
B~ substituting 1-(2-mercaptoacetyl)-L-azetidine-2- -
carboxylic acid for the 3-mercaptopropanoyl-L-proline in the
procedure of Example 110, l,ll-(dithiobisacetyl)-bis-L- ,
azetidine-2~carboxylic acid is obtained.
-58-
Example 115
..
1,1'-[Di-thiobis(3-propanoyl)]-bis-L-pipecolic acid
By substituting 3-mercaptopropanoyl-L-pipecolic acid
for the 3-mercaptopropanoyl-L-proline in the procedure of
Example 110, 1,1'-[dithiobis(3-propanoyl)]-bis-L-pipecolic
acid is obtained. '
E~ample 116
1,1'-[Dithiobis(3-propanoyl)]-bis-4-methyl-L-prolihe ;
By substituting 1-(3-mercaptopropanoyl)-4-methyl-I,-proline
for the 3-mercaptopropanoyl-L-proline in the procedure of Example
110~ 1,1'-[dithiobis(3-propanoyl)]-bis-4-methyl-L-proline is
obtained.
Example 117
1,1'-~Dithiobis(3-propanoyl)]-bis-5-hydroxy-L-pipecolic acid ~
" .~ .
By substituting 1-(3-mercaptopropanoyl)-5-hydroxy-L~
pipecolic acid for the 3-mercaptopropanoyl-L-proline in the pro~ ~ ~,
cedure of Example 110, 1,1'-[dithiobis(3-propanoyl)]-bis-5-hydroxy ' ,;
L-pipecolic acid is obtained. ~ ,
Example 118 ,~
1,1'-~Dithiobis(2-benzyl-3-propanoyl)]-bis-L-proline
By substituting 1-(3-mercapto 2-benzylpropanoyl)-L-proline
for the 3-mercaptopropanoyl-L-proline in the procedure of ,Example ',
110l l,l'-[dithiobis(2-benzyl-3-propanoyl)]-bis-L-proline is ;'
obtained. '
~', '
-59-
EY~ample 119
1,1'-~Dithiobis)2-methyl-3-propanoyl)]-bis-I,-pipecolic acid
By substituting 1-(3-mercapto-2-methylpropanoyl)-I,-
pipecolic acid for the 3-mercaptopropanoyl-L--proline in the
procedure of Example 110, 1,1'-[dithiobis(2-methyl 2-propanoyl)}-
bis-L-pipecolic acid is obtained. ;
Example 120
1,1'-~Dithiobis)4-butanoyl)~-bis-L-proline
By substituting 4-mercaptobutanoyl-L-proline for the
3-mercaptopropanoyl-L-proline in the procedure of Example
110, 1,1'-~dithiobis(4-butanoyl)]-bis-L-proline is obtained.
Example 121
1,1'-[Dithiobis(2-benzyl-4-butanoyl)}-bis-L-proline
By substituting 1-(4-mereapto-2-benzylbutanoyl)-L-proline
for the 3-mereaptopropanoyl-L-proline in the procedure o~
Example 110, 1,1'-[dithiobis(2-benzyl-4-butanoyl)]-bis-L-proline
is obtained.
Example 122
1,1l-[Dithiobis(3-butanoyl)]-bis-L-proline ~'
.
By substituting 3-mercaptobutanoyl-L-proline for the ~ ;'
3-mereaptopropanoyl-L-proline in the proeedure of Example
110, 1,l'~[dithiobis(3-butanoyl)]-bis-L-proline is obtained. ~-
-60
6~ :
Example 123
1,1'-[Dithiobis(3-propanoyl)]-bis-L-proline methyl ester
A solution of l,l'-[dithiobis(3-propanoyl)l-bis-L-
proline in methanol is treated with ethereal diazomethane ;
until persistent yellow color~ After fifteen minutes a
few drops of acetic acid are added and the solvents are .
removed in vacuo to yield, l,l'-[dithiobis(3 propanoyl)l-
-
bis-L-proline methyl ester.
Example 124
l,l'-[Dithiobis(3-propanovl)]-bis-L-proline amide
A solution of 1,1'-[dithiobis(3-propanoyl)]-bis-
L-proline methyl ester in methanol is saturated with am- . :
monia while cooling in an ice-water bath. The reaction ~:
mixture is stored for 16 hours at room temperature in a
pressure bottle, and then the solvents are removed ln . ~ .
vacuo to yield l,l'-[dithiobis(3-propanoyl)]-bis-L-proline
amide. ~ ~.
3~
1,1'-[Dlthiobis(2-phenyl-3-propanoyl)]-bis-L-proline
:::
By substituting 1-(3-mercapto-2-phenylpropanoyl)-
L-proline for the 3-mercaptopropanoyl-L-proline in the
procedure of Example llO, l,l'-[dithiobis(2-phenyl-3-pro-
panoyl)]-bis-L-proline is obtained.
-61-
.. . ', ;,.
P3~4~
Example 126
1,1'-[(Sulfinylthio)-bis-(3-propanoyl)]-bis--L-proline
.
While cooling in an ice bath 0.12 mo:Le of peracetic
acid is added to a stirred solution o~ l,l'--[dithiobis(3-
propanoyl)]-bis-L-proline (40 g) in glacial acetic acid
(500 ml). The reaction mixture is allowed to stand over-
night at room temperature and the solvent is then removed
ln vacuo to ~ield l,l'-[(sulfinylthio)-bis-(3-propanoyl)]-
bis-L-proline.
Example 127
1,1'-[(Sulfon~lthio)-bis-(3-propanoyl)]-bis-L-prollne
A 30% solution of hydrogen peroxide (2.0 ml) is
added to a solution of l,l'-Edithiobis(3-propanoyl)]-bis-
L-proline (4 g) in glacial acetlc acid (80 ml) and the
solution is stored for thirty hours at room temperature.
The solvent is removed ln vacuo to yield l,l'-[(sulfonyl-
thio)-bis-(3-propanoyl)]-bis-L-proline.
Example 128 -
.
1,1'-[(Sulfinylthio)-bis-(2-propanoyl)]-bis~L-proline --
By substituting 1,1'-[dithiobis(2-propanoyl)]-
bis-L-proline for the 1,1'-[dithiobis(3-propanoyl)]-bis-
L-proline in the procedure of Example 126, l,l'-[(sul-
finylthio)-bis-(2-propanoyl)]-bis-L-proline is obtained.
-62-
6~
Example 129
1,1'-[(Sul~inylthlo)-bis-acetyl]-bis-L-azetidine-2-carbox -
Y .
lic acid
By substitut.ing l,l'-(dithiobisacetyl)-bis-L-azeti-
dine ca.rboxylic acid for the l,l'-[dithiobis(3-propanoyl)]-
bis-L-proline in the procedure of Example 126, l,l'-[(sul-
finylthio)-bis-acetyl]-bis-L-azetidine-2-carboxylic acid
is obtained.
Example 130 ~:
l,l'-[(Sul~inylthio) bis-(3-propanoyl)]-bis-4-methyl-L-
proline -.
.
By substituting 1,1'-[dithiobis(3-propanoyl)]-bis- ~; :
4-methyl-L-proline for the 1,1'-[dithiobis(3-propanoyl)]-
bis-L-proline in the procedure of Example 126, l,l'-[~sul-
finylthio)-bis-(3-propanoyl)]-bis-4-methyl-L-proline is . :-
obtained.
Example 131 ::~
1,1'-[(Sulfinylthio)-bis-(2-benzyl-3-pro_ noyl)]-bis-L-
proline
By substituting 1,1'-[dithiobis(2-benzyl-3~propa-
noyl)]-bis-L-proline for the 1,1'-~dithiobis(3-propanoyl)]-
bis-L-proline in the procedure of Example 126, l,l'-[(sul-
finylthio)-bis-t2-benzyl-3-propanoyl)]-bis-L-proline is
obtained.
Example 132
1,1'-~(Sulfinylthio)-bls-(4-butanoyl)~-bis-I.-proline
By substitu-ting 1,1'-[dithiobis(4-butanoyl)]-bis-
L-proline for the 1,1'-[dithiobis(3--propanoyl)]-bis-L-
proline in the procedure of Example 126, l,l'-~(sulfinyl--
thio)-bis-(4-butanoyl)]~bis-L-proline is obtained.
-63-
;9L
Example 133
1 l'-[(Sulfin l-thio)-bis-(3-butano l)]-bis-L-proline
,, Y Y
By subs-tituting 1,1'-[dithiobis(3-bu-tanoyl)]-bis-L-
prol.ine for the l,l'-[dithiobis(3-propanoyl)~-bis-L-proline
in the procedure of Example 126, l,l'-[(sulfinyl-thio~-bis-
(3-butanoyl)]-bis-L-proline is obtained.
Example 134
1,1'-[(Sulfinyl-thio)-bis-~2-methyl-3-propanoyl)-bis-L-pro-
line
By substituting 1,1'-[dithiobis(2-methyl-3-propan~
oyl)]-bis-L-proline for the 1,1'-[dithiobis(3-propanoyl)]-
bis-L-proline in the procedure of Example 126, 1ll'-[(sul-
finylthio)-bis-(2-methyl-3-propanoyl)~-bis-h-proline is
obtained.
,
Example 135 :
1,1'-~(Sulfinylthio)-bis-(2-phenyl-3-propanoyl)]-bis-L-
proline
By substituting 1,1'-[dithiobis(2-phenyl-3-propa-
noyl)l-bis-L-proline for the 1,1'-[dithiobis(3-propanoyl)]- .
bis-L~proline in the procedure of Example 126, l,l'-[(sul-
finylthio)-bis-(2-phenyl-3-propanoyl)]-bis-L-proline is ~ :
obtained. :`
Example 136
1-[3-1[3-(2-Carboxy-l-pyrrolidinyl)-3-oxopropyl]-dithio]-
2-methylpropanoyl]-L-proline
By substituting 1,1'-1~sulfinylthio)-bis-(2-methyl-
3-propanoyl)]-bis-L proline for the ethylthiosulfinate in
the procedure of Example 97, 1-[3-[13-(2-carboxy-1-pyrro-
lidinyl)-3-oxopropyl]dithio]-2-methylpropanoyl]-L-proline
is obtained.
-64-
~ /~
36~L
Example 137
l,l'-[(Sulfonyl-thio)-bis-acetyl)-bis-L-hydroxyproline
sy substituting l,l'-(dithiobisacety:L)-bis-L-hy-
droxy proline ~or the l,l'-[dithiobis(3-propanoyl)-bis-L-
proline in -the procedure of Example 127, l,l'-[(sulfonyl-
thio)-bis-acetyl)-bis-L-hydroxyproline is obtained~
Example 138 ~ ;
1,1'-[(Sulfonylthio)~bis~(3-propanoyl)]-bis-L-pipecolic
acid
By substituting 1,1'-[dithiobis(3-propanoyl)]-
bis-L-pipecolic acid for the 1,1'-[dithiobis(3-propanoyl~]-
bis-L-proline in the procedure of Example 127, l,l'-[~sul-
fonylthio)-bis-(3-propanoyl)]-bis-L-pipecolic acid is ob-
tained.
Example 139
1,1'-[(Sulfonylthio)-b_s-(3-propanoyl)]-bis-5-hydroxy-L-
pipecolic acid ~ ~
: ' ,
By substituting 1,1'-[dithiobis(3-propanoyl)]-bis- ;
5-hydroxy-L-pipecolic acid for the 1,1'-[dithiobis-(3- ;
propanoyl)]-bis-L-proline in the procedure of Example 127,
1,1'-[(sulfonylthio)-bis-(3-propanoyl)]-bis-5-hydroxy-L-
pipecolic acid is obtained.
Example 140
1,1'-[(Sulfonylthio)-bls-(2-methyl-3-propanoyl)]-bis-L-
pipecolic acid
:
By substituting 1,1'-[dithiobis(2-methyl-3-propa-
noyl)]-bis-L-pipecolic acid for the 1,1'-[dithiobis(3-
propanoyl)]-bis-L-proline in the procedure of Example 127,
1,1'-[(sulfonylthio)-bis-(2-methyl-3-propanoyl)]-bis-L-
pipecolic acid is obtained.
-65-
': :
E_ mple 141
l,l'-[(Sulfonylthio)-bls-(2-ben2yl-4-butanoyl)]-bis-L~
proline
By subs-titu-ting 1,1'-[di-thiobis(2-benzyl-4-bu-tan-
onyl)]-bis-L-proline for the 1,1'-[dithiobis(3-propanoyl)]-
bis-L-proline in the procedure of Example 127, l,l'-l(sul-
fonylthio)-bis-(2-benzyl-4-butanoyl)]-bis-L-proline is ob-
tained.
Example 142
3-Acetylthio-2-phenylpropanoic acid
By substi-tuting 2-phenylacrylic acid for the meth-
acrylic acid in -the procedure of Example 25, 3-acetylthio-
2-phenylpropanoic acid is obtained.
1-(3-Acetylthio-2-phenylpropanoyl)-L-proline tert-butyl
ester
By substituting 3-acetylthio-2-phenylpropanoic acid
for the 3-acetylthio-2-methylpropanoic acid in the proce-
dure of ExampIe 28, 1-(3-acetylthio-2-phenylpropanoyl3-L-
proline tert-butyl ester is obtained.
Example 143
1-(3-Mercapto-2-phenylpropanoyl)-L-proline
By substituting 1-(3-acetylthio-2 phenylpropanoyl)-
L-proline tert-butyl ester for the 1-(3-acetylthio-2-
me-thyl-propanoyl-L-proline tert-butyl ester in the pro-
cedure of Example 29, and subjecting the product to am-
monolysis as in Example 34, 1-(3-acetylthio-2-phenylpro-
panoyl)-L-proline and 1-(3-mercapto-2-phenylpropanoyl)-L-
proline are obtained.
.:
-66-
36~
HA135b
Example 144
1-[3-(Acetylthio)-DL-propanoyl]pipecolic acid
Pipecolic acid (6.5 g.) is suspended in 200 ml.
of dimethylacetamide. 3-acetylthiopropanoyl chlorlde
(8.3 g.) is added dropwise at 23 to the suspension. A
clear solution forms and the temperature rises to 28 .
To this clear solution is added N-methylmorpholine (1~.1 g.).
An immediate precipitate forms and the temperature rises
to 34 . The mixture is heated on a steam bath for 1 hour
when a clear solution forms. Qn cooling, the precipitated
solid is filtered to yield 5.1 g. of 1-[3-(ace-tylthio)-DL-
propanoyl]pipecolic acid, m.p. 190-200. The solvent is
removed and the viscous residue is triturated with isopropyl
ether to yield 7.8 y. of product, m.p. 98-101 . Recrystalliza- ;~
tion from acetone-hexane yields d constant melting solid,
m.p. 102-104 ; Rf 0.72 [silica gel, benzene, acetic acid~(7~:2)].
Example 145
DL-1-(3-Mercaptopropanoyl)pipecolic acid
12 ml. of concentrated ammonium hydroxide is
stirred under nitrogen at 10 for about l5 minutes, then
solid 1-[3-tacetylthio)-DL-propanoyl]-pipecolic acid (6.6 g.)
is added at 5 to 10 . A clear solution forms after 2-3
minutes. The ice bath is removed and the solution lS stirred
at room temperature under nltrogen for 45 minutes. The
solution ls made strongly acid with 20~ HCl ~cooling) and
the precipitated oil is extracted with 3 x 150 ml. of ethyl
acetate. The ethyl acetate extracts are dried over magnesium
sulfate and the solvent is removed to yield 6.0 g. of
DL-1-(3-mercaptopropanoyl)pipccolic acid, Rf 0.77 [sllica gel,
3d benzene,acctic acid (7:1)].
-~,7-
HA135b
Example 146
1-(3-Mercaptopropanoyl)-L-pipecolic acid
By substituting L-pipecolic acid Eor the DL-
pipecolic acid in the procedure of Example 144 and then
submitting the product to the procedure of Example 145,
1-[3-(acetylthio)propanoyl]-L-pipecolic acid and 1-(3-
mercaptopropanoyl)-L-pipecolic acid Rf 0.80 [silica gel,
benzene, acetic acid (7:1)], [a] -51.5 (c, 1.0 abs.
ethanol), are obtained.
Example 147
1~[3-(Acetylthio)-2-methylpropanoyl-DL-pipecollc acid
6.5 g. (0.05 m.) of pipecolic acid are suspended
in dimethylacetamide (200 mg.), 9.0 g. (0.05 m.) of
3-acetylthio-2-methylpropanoyl chloride is added dropwise.
The temperature rises to 29 and a clear solution forms
Then 10.1 g. of N-methylmorpholine is added a]l at once
and the temperature rises to 34~. The mixture is heated
on a steam bath for 1 hour when a clear solution forms.
This is allowed tP stand at room temperature overnight
and the solid which precipitates is filtered to yield
6.1 g.j m.p~ 203-204 . The solvent is removed and the
viscous residue is triturated with water and 20% HCl. The
yellow oil is extracted with 3 x 150 ml. of ethyl acetate.
~he ethyl acetate extracts are dried over magnesium sulfate
and removed to yield 14 g. of 1-[3-(acetylthio)-2-methyl-
propanoyl-DL-pipecolic acid as a viscous oil.
Example 148
1-(3-Merca~to-2-methylpro~anoyl)-DL~pipecolic acid
~ .
Aqueous NH4011 (30 ml. water and 20 ml. conc. NH40H)
is stirred under nitro~en a~ 10 for 15 minutes. This i9
-6~-
~ ~.
~ .
HA135b
added to 13.0 g. (0.05 m) of 1-[3-(acetylthio)-2-
methylpropanoyl]-DL-pipecolic acid and -the resulting solution
is stirred for 10 minutes under nitroyen; then at room
temperature for 50 minutes. It is then treated with water ~;
and 20~ HCl and the yellow oil extracted with 3 x 150 ml.
of ethyl acetate. The ethyl acetate extract is dried over - -
magnesium sulfate and removed to yield 11.1 g. 1-(3-
mercapto-2-methylpropanoyl)-DL-pipecolic acid as a viscous
oil. R~ 0.62 [silica gel, benzene, acetic acid (7:2)].
Example 149
3-[(4-Methoxyphenyl)methylthio]--2-methylpropanoic acid
p-Methoxy-~-toluene thiol (15.4 g., 0.1 mol.) is
added to a solution of methacrylic acid (8.6 g., 0.1 mol.)
in 50 ml. 2~ sodium hydroxide. The mixture is heated on
the steam bath ~or three hours, then refluxed for two
:
hours and cooled. The mixture is~extracted with ether, then
the aqueous layer is acidifled with concentrated HCl and
extracted with dichloromethane. The acidic extracts are
washed wlth brine, dried (MgSO4) and evaporated in~vacuo.
2n The resulting semi-solid is taken up in 50 ml. of dlchloro-
methane, diluted with 50 ml. hexane, and chilled. 3-[(4
methoxyphenyl)methylthio]-2-methylpropanoic acid is
collected as a white crystalline solid, m.p. 74-82
~5.5 g.).
Example 150
1-[3-(4-Methoxyphenyl?methylthio~-2-met
proline tert-butyl ester ~
3-~(4-methoxyphenyl)methylthio]-2-methylpropanoic
acid (3.6 g., 0.015 mol.~, L-proline tert-butyl ester
(2.6 g., 0.015 mol.), and dicyclohexylcarbodiimide (3.1 g.,
-69-
.
i~l864 HA135b
0.015 mol.~ are dissolved in 50 m:L. oE dichloromethane
and stirred thirty minu-tes at 0 . The cooling bath is
removed and the mixture stirred overnight (sixteen hours).
The resulting suspension is filtered and the filtrate
washed with 5~ potassium bisulfate, salurated sodium bicarbonate
and brine, then dried (MgSO4~ ancl evaporated in vacuo.
The resulting clear oil is applied to a 250 ml. silica
gel column and chromatographed using 20% ethyl acetate/
hexane as eluant. The main fraction (Rf = 0.70, silica
gel, ethyl acetate) is evaporated to 5.5 g. (g3~) of 1-
[3-(4-methoxyphenyl)methyl-thio3-2-methylpropanoyl-L-proline
tert-butyl ester as a clear oil. Rf = 0.70 (silica gel,
ethyl acetate); Rf = 0.60 (silica gel, ether).
Exam~le 151
1- ~ 2-methylpropanoyl)-L-proline
The ester from Example 150 (1.2 g., 0.003 mol.),
anisole (5 ml.) and trifluoromethanesulfonic acid (0.5~ml.)
are dissolved in 20 ml. of -trifluoroacetic acid under
nitrogen, and the resultinq red solution let stand one hour
at room temperature. The solution is evaporated in vacuo
to a red residue which is taken up in ethyl acetate and
washed with water! brine, then dried (MgSO4) and evaporated.
The residue is repeatedly triturated with hexane and the
residual hexane evaporated; the oil residue amounts to
0.4 g~ A portion (180 mg.) of this material ig subjected
to preparative thin-layer chromatography on 2 mm silica gel
plates using benzenejacetic acid 75:25 as eluant. The
main nitroprusside-positive band (Rf = 0.40) is recovered,
affording 135 mg. of 1-(3-mercapto-2-méthylpropanoyl)-L-
proline as an oil. TLC using ben~ene/ace-tic acid 75:25
-70-
:
HA135b
(R = 0 40) and chloroform/methanol/acetic acid 50:40:10
(Rf = 0.62).
Example 152
1-(3-Mercapto-2-D-methylpropanoyl)-L-proline
IJnder a blanket of argon 1-[3-(acetylthio)-2-
~methylpropanoyl]-L-proline (10.0 g.) is slurried in
water (150 ml.) at 10 . To this mixture is added 5N
sodium hydroxide and the pH of the solution maintained
at 13 for 1.5 hours. After this time, when the uptake
of sodium hydroxide had ceased, the solution is
acidified to a pH = 2.0 with concentrated sulEuric acid.
The aqueous solution is then extracted three
times with methylene chloride ~3 x 150 ml.) and the
combined methylene chloride fractions concentrated to
an oil. The concentrate is taken up in ethyl acetate,
filtered and the filtrate diluted with hexane (30 ml.).
: -
An additional amount Oe hexane is added after 1/2 hour
and then the mixture cooled to 10 for 1 hour.
The crystals are filtered and washed with hexane
(2 x 25 ml.) and dried to constant weight to give
1-(3-mercapto-2-D methylpropanoyl)-L-proline as white
crystals, 6.26 g., m.p. 100-102.
Example 153
.. . ~ ~ :
1-~3-Tosyloxy-2-methylpropanoyl]-L-proline
By substituting 3-tosyloxy-2-methylpropanoic acid
chloride for the 3-acetylthio-2-methylpropanoic
acid chloride in the procedure of Example 29b 1-[3-
tos~loxy-2-methylpropanoyl]~L-proliAe is obtained.
., .
-~71-
- ' .
. . ' . ~ : . ~' ,''
~1~1864 i~A135b
Example 154
1-L~ Y_thio-2-methylpropanoyl]-L-proline
1-[3-Tosyloxy-2-methylpropanoyl~-L-proline
(3.5 g.) is added to a solution of thiolacetlc acid
~1.14 g.), and triethylamine (3.5 ml.) in ethyl acetate
(20 ml.). The solution is maintained at 50 for three
hours, cooled, diluted with ethyl acetate (100 ml.), and
washed with dilute hydrochloric acid. ~he organic layer
is dried and concentrated to dryness in vacuo. The
residue is dissolved in acetonitrile and dicyclohexylamine
is added. The crystalline precipitate is recrystallized -~
from isopropanol to yield l-[3-acetylthio-2-D-methyl-
propanoyl)-L-proline, dicyclohexylamine salt, m.p.
187-188 , Ea]D -67 (c 1,4, EtOH). This salt is
converted to the free acidj m.p. 83-85 (an isomorphic
form of m.p. 104-105 is obtained if the crystallizing
solution is seeded wi-th high melting material).
Example 155
1-~3-Mercaptopropanoyl)-L-proline, t-butyl ester
To a stirred solution of 1.71 g. (10 mmoles) of
proline t-butyl ester and 1.35 g. (l0 mmoles) of 1-
hydroxybenzotriazole hydrate in 20 ml. of N,N-dimethyl-
formamide at 0-5 are added 2.06 g. (10 mmole) of N,N'-
dicyclohexylcarbodiimide. The mixture is stirred for
10 minutes, followed by the addition of 1.06 g. (10 mmole)
of 3~mercaptopropanoic acid in 2 ml. of N,N-dimethyl-
formamide- The mixture is then stirred at 0-5 for
1 hour, and at room -temperature overnlght. ~
The precipitated N,N'-dicyclohexylurea is
filtered off, and the filtrate concentrated in vacuo. ~-
-72~
~ .
4 HA135b
The residue is taken up in ethyl ace-ta-te, washed
thoroughly with saturated aqueous sodi~lm bicarbona-te,
dried, and concen-trated in vacuo to 2.5 g. of oil.
The oil is taken up in 1:1 ethyl acetate-hexane
and applied to a silica gel column (100 g.). Elution
with 1:1 ethyl acetate-hexane aEfords 1.40 g. (54~) of
1~(3-mercaptopropanoyl)-L-proline/ t-butyl ester as an
oil, which crystallizes on standing. Recrystallization
from ether-hexane yields 0.9 g. of colorless crystalline
solid, m.p. 55-60, identical to the compound of
Example 17.
Example 156
1-(3-Mercaptop _panoyl)-L-proline
A solution of 75 mg. (0.27 mmole) of 1-[3-[[(ethyl-
amino)earbonyl]thio]propanoyl]-L-proline in 1 ml. eaeh of
coneentrated ammonium hydroxide anù water is allowed to
stand at room temperature for 18 hours under argon~ The~
`solution is diluted with a small amount of water~and
extraeted with ether. The aqueous layer is acidified with
eold coneentrated hydrochloric acid and extraeted with
ethyl acetate. The combined extracts are dried and
eoncentrated in vacuo to give a eompound identical with
the product of Example 18. TLC (silica gel; benzene:acetic
acld 7:3) Rf 0.~.
Example 157
Methaeryloyl-L-Proline
L-preline (23.0 g., 0.2 mol.) is dissolved in
100 ml. water and stirred in an iee bath. ~ethacryloyl
.
- ehloride (19.6 ml., 0.2 mol.~ in 25 ml. of methyl isobutyl
ketone is added dropwise over three hours. Sedium hydroxlde
'~
HA135b
solution (2N) is added simultaneously, maintaining the
pH of ~he reaction mixture at 7Ø Addition of base is
continued for four hours after addition of acid chloride
has been completed. The reaction mixture is adjusted to
pH 5 with concentrated HCl and extracted with ethyl
acetate. The aqueous layer is then acidified to pH 2.5
and extracted thorouyhly with ethyl acetate. The acidic
extracts are washed with brine and drled (MgSO4). The
ethyl acetate solution is trea-ted with dicyclohexylamine
(40 ml.) and chilled overnight. The resulting white pre-
cipitate is filtered and dried, yielding 29 g. (39~) of `~
white solid, m.p. 202-210 . The solid is crystallized from
1.5 liters 3:1 acetonitrile/isopropanol to yield 19.7 g.
of methacryloyl-L-proline, dicyclohexylamine salt as
fine white needles, m.p. 202-210 .
The salt is dissolved in water/ethyl acetate
and the mixture acidified with concnetrated HCl. The
resulting suspension is filtered to remove a fine white
precipitate which is washed well with ethyl acatate. The
filtrate is saturated with sodium chloride and extracted
thoroughly with ethyl acetate. The extracts are washed
with brine, dried (MgSO4) and evaporated to a clear oil
which solidifies. Crystallization Erom ethyl acetate/hexane
yields 7.5 g. (83%) of methacryloyl-L-proline as a
white crystalline solidr m.p. 89-93 . An analytical
sample is obtained by recrystallization, m.p. 95-98.
Example 158
~.
1-(3-Acetylthio-2-D-methylpropanoyl)-L-proline
Methacyloyl-L-proline (183 mg., 0.001 mol.) is
dissol~ed in thiolace~ic aci(l (0.5 ml.) an~ allowe~l
-7~-
HA135b
to stand at room temperature for sixteen hours. The
solution is evaporated ln vacuo to a yellow residue.
Preparative thin layer chromatography (silica gel, di-
chloromethane/methanol/acetic acid 90:5:5) allows isolation
of a clear oil (240 mg.) as -the main fraction. TLC (dichloro-
methane/methanol/acetic acid 90:5:5) shows ~his material
to be l-(3-acetyl-thio-2-DL-methylpropanoyl)-L-proline
corresponding to the product of Example 29B. Rf = 0.35;
(benzene/acetic acid 75:25) Rf = 0.38.
The oil is dissolved in 3 ml. acetonitrile,
treated wi-th dicyclohexylamine until the solution is
basic, and chilled. A white crystalline solid (106 mg.)
m.p. 175-181 , is collected. Crystallization from
isopropanol gives l-(3-acetylthio-2-D-methylpropanoyl)-
L-proline, dicyclohexylamine salt/ rn.p. 187-188 ,
identical with this product in Example 29A.
Example 159
l-[Dithiobis-(2-methyl-3~propanoyl)]-bis-L-proline
By substituting 3,3'-dithiobis-2-methylpropanoic acid
for the 3-ace~tylthio-2-methylpropanoic acid ln the pîO- :
cedure of Example 29B, l-[dithiobis-t2-methyl-3-propanoyl)]-
bls-L-proline is obtained.
Exam~le 160
1-(3-Mercapto-2 - methylpropanoyl)-L-proline
Zinc dust (10.0 g.) is added to a slurxy of the
product of Example 159 (5~0 g.) in 100 ml. of 1 O N
sulf~uric acid and the mixture is stirred at 18 for four
hours under a blanket of nitrogen. ~he solution is then
filtered, the Zinc washed with wat~r (20 ml.) and the
combined filtrates are ~tracted with methylene chloride
-75~
HA135b
(3 x 75 ml~). The methylene chloride washes are back
extracted with water (25 ml.) and then the organic
solution concentrated to an oil. This oil is taken up
in ethyl acetate (20 ml.) and filtered. Hexane ~15 ml.)
is added to the filtrate and the mixture is stirred for
15 minutes. After this time, an additional volume of
hexane (30 ml.) is added and the solution cooled to 5
for 1 hour. The mixture is then fil~ered, and the product
is washed with hexane (2 x 10 ml.) and dried to give 4.17 g.
of white crystals o~ the product, 1-(3-mercapto-2-
methylpropanoyl~-L-proline. TLC, Rf = 0.60
~Solvent system: benzene/acetic acid 75:25).
Example 161
3-Benzylthio-2-methylpropanoic acid
By substituting a-toluenethiol for p-methoxy a-
toluenethiol in the procedure of Example 149, 3~benzylthio-2-
methylpropanoiC acid is obtained.
Example 162
1-[3-(Benz~lthio)-2-methylpropanoyl]-L-proline tert.
bu-tyl ester
By sub~ti-tuting 3-benzylthio-2-methylpropanoic ~`
acid for the 3-[(4-methoxyphenyl)methylthio]-2-methyl-
propanoic acid in the procedure of Example 150, 1-[3-
:
~benzylthio)-2-methylpropanoyl]-L-proline tert. butyl ester
is obtained.
Example 163
1=[3-(Benzylthio)-2-methylpropanoylJ-~-proline
1-[3-(benzylthio)i-2-methylpropanoyl]-~-proline
tert. butyl ester (7.8 g.) is dissolved in a mixture of
anisole (55 ml.) and trif:luoroacetic acid (110 ml.~. Afier
-76-
HA135b
one hour storage at room temperature, the solvent is
removed in vacuo and the residue is dissolved in ether,
washed several times with saturated sodium chloride, dried
over magnesium sulfate and evaporated to dryness in vacuo
to yield 1-~3-(benzylthio)-2-methylpropanoyl}-L-proline.
Rf 0.5 (Silica gel~ Benzene/acetic acid 3:1) Rf 0.5.
(Silica gel, Methyl-ethylketone/acetic acid/pyridine/water
14:1:2:1).
Example 164
1-(3-Mercapto-2-methylpropanoyl)-L-proline
1-[3-(benzylthio)-2-methylpropanoyl]-L-proline
(0.1 g.) is suspended in boiling liquid ammonia (10 ml.)
and small pieces of sodium are added with stirring until
persistent blue color. The color is discharged with a
few crystals of ammonium sulfate and the ammonia is~allowed
to evaporate under a current of nitrogen. The residue is
dissolved in a mixture of dilute hydrochloric acid and ethyl
acetate. The organic layer is dried and concentrated to
dryness in vacuo to yield l-(3-mercapto-2-methylpropanoyl)-
L-proline. R~: 0.35 (Silica gel; Benzene/acetic acid
3!1), Rf 0.5 (Sillca gel; Methyl-ethylketone/acetic acld~ -
pyridine/water ~ 2:1? identical to the compound of
Example 34.
Example 165
3-Triphenylmethylthio-2-methylpropanoic acid
A solution of 3-mercapto-2-methylpropanoic acld
(1.2 g.)~and tritylchloride (2.9 g.) in me~hylene
chloride (50 ml.) is kept at room temperature for 2 hours.
~The mixture is warmed in a steam bath for 20 minutes and then
evaporated to dryness in vacuo and -he residue is d1ssolved
HA135b
in saturated aqueous sodium bicarbonate and the solution
is washed with ethyl acetate. The aqueous phase is
acidified to pH 3 and extracted with ethyl acetate. The
organic layer is dried and concentrated to dryness to give
3-triphenylmethylthio-2-methylpropanoic acid. ~f 0.8
(Silica gel, BenzeneJace-tic acid 3:1).
Example 166
1-[3-(Triphenylmethylthio)-2-methylpropanoyl]-L-proline
tert.butyl ester
By substituting 3-triphenylmethylthio-2-methyl-
propanoic acid ~or the 3-[(4-methoxyphenyl)methylthio]-
2-methylpropanoic acid in the procedure of Example 150,
1-[3-(triphenylmethylthio)-2-methylpropanoyl]-L-proline
tert.butyl ester is obtained.
Example 167
1-[3-(Triphenylmethylthio)-2-methylpropanoyl]-L-proline
3-Triphenylmethylthlo-2-methylpropanoic acid
(1.8 g.) and N,N'-carbonyldiimidazole t0.8 g.) are~
dissolved in tetrahydrofuran (10 ml.) with stirring at ~ ~-
room temperature. After twenty minutes, the solution is
added to a mixture of L-proline (0.6 g.) and N-methyl-
morpholine (I g~) in dimethylacetamide (20 ml.) The
resulting mixture is stirred overnight at room temperature,
çoncentrated ta dryness and the residue dissolved in a
mixture of ethyl acetate and lQ% aqueous potassium bisulfate.
The orga~ic layer is separated and dried and concentrated
t~ dryness in vac:uo to obtain 1-[3-(triphenylmethylthio)-
2-methylpropanoyl~-L-proline Rf: = 0.4 (~ilica gel,
Benzene/acetic acid 3:1), Rf 1~0 (Silica gel, Methyl-ethyl-
ketone/acetic acid/pyridine/water 14:1:2
-78-
~ HA135b
Exarnple 168
1-[3-Mercapto-2-methylpropanoyl)-L-proline
1-[3-(triphenylmethylthio)-2-methylpropanoyl]-L-
proline tert.butyl ester ~5 g.) is dissolved in a mixture
of anisole (55 ml.) and trifluoroacetic acid (110 ml.).
A~ter one hour storaye at room temperature, the solvents
are removed in vacuo and the residue i5 applied to a
column of silica gel equilibrated with benzene:acetic
acid (75:25) and eluted with the same solvent. The
fractions corresponding to the component with Rf 0.40
(TLC silica gel with same system) are pooled and concentrated
to dryness to yield l-[3-mercapto-2-methylpropanoyl)-L-
proline. Rf 0.62 (silica gel, chloroform/methanol:
acetic acid:water 50:40:10), identical to the compound
of Example 34.
Example 169
3-(Tetrahydropyran-2-ylthio)-2-methylpropanoic acid
To a solution of 3-mercapto-2-methylprop noi~c
acid (2.4 g.) and freshly distillled 2,3-dihydro-4EI-
pyrane (1.9 g.) in benzene (60 ml.), boron trifluoride
etherate (2.8 g.) is added. After two hours, potassium
carbonate (4 g.) is added, the mixture is stirred and
filtered. The iltrate is concentrated to dryness to~
yield 3-(tetrahydropyran-2-ylthio)-2-methylpropanoic acid.
Example _ 0
1-~3-(Tetrahydropyran-2-ylthio)-2-methylpropanoyl]-L-proline
By substituting 3 (tetrahydropyran-2-ylthio)-2-
methylpropanolc acid for the 3-triphenylmethylthio-2-
methylpropanoic acid in the procedure of ~xample 167,
1-[3-(tetrahydropyran-2-ylthio)-2-methylpropanoyl]-L-proline
-79-
HA135b `
is obtained. Rf: 0.$ (Silica gel, Benzene/acetic acid
3:1; Rf: 0.75 (Silica gel, Methyl-ethylketone/Acetic acid/
pyridine/water; 14:1:2:1).
Example 171
1-(3-Mercapto-2-methylpropanoyl~-L-proline
A solution of 1-~3-(tetrahydroPyran-2-ylthio)~2-
methylpropanoyl)-L-proline (1 g.) in a mixture of methanol
(25 ml.) and concentrated hydrochloric acid (25 ml.) is
stored at room temperature for 30 minutes. The solvents
are removed in vacuo to yield 1-(3-mercapto-2-methyl-
propanoyl~-L-proline. ~: 0.35 (silica gel, Benzene/
acetic acid, 3:1), Rf 0.5 (silica gel, Methyl-ethylketone/
acetic acid/pyridine/water 14:1:2:1) identical to the
compound of Example 34.
Example 172
3-Acetamidomethylthio-2-methylp~opanoic acid
3-Mercapto-2-methylpropanoic acid (2.4 g~) and
N-hydroxymethylasetamide (1.3 g.) are dissolved in
tri~luoroacetic acid and the solution is stored at room
temperature for one hour. The trifluoroacetic acid is
removed in vacuo and the residue is dried in vacuo over
potassium hydroxide to yield 3~acetamidomethylthio-2-
methylpropanoic acid.
Example 173
1-[3-(Acetamidomethylthio)-2-methylpxopanoyl]-L-proline
By substituting 3~acetamidomethylthio-2-methyl-
propanoic acid for the 3-(tetrahydropyran-2-yl hio)-2-
methylpropanoic acid in the procedure of Example 170
1-[3-(ace~amido~ethylthio~2-
--~0--
.:
10 ~ 4 HA13Sb
methylpropanoyl]-L-proline is obtained. Rf 0.2 (Sllica
gel, Benzene/acetic acid 3:1) R 0 3 tSilica gelr Methyl-
ethylketon~/acetic acid/pyridine/water 14:1:2
Example 174
1-(3-Mercapto-2-methylpropanoyl ? -L-proline
1-[3-(acetamidomethylthio)-2-methylpropanoyl~-L-
proline (1.4 g.) and mercuric acetate (1.93 g.) are dissolved
in a mixture of acetic acid (25 ml.) and water (25 ml.).
After one hour stirring on the steam bath, hydrogen sulfide
is bubbled through until no more precipitation of~mercuric ~ -
sulfide i5 observed. The mixture is filterd, the precipitate
is washed with ethanol, and the filtrate is concentrated
to dryness in vacuo to ~ield l-(3-mercapto-2-rnethylpropanoyl)-
L-proline. Rf: 0.35 ~Silica gel, Benzene/Acetic acid 3:1);
Rf : 0.5 ~Silica gel, Methyl-ethylketone~Acetic acid/
pyridine/water 14:1:2:1) identical to the compound of ~ ~`
Example 34.
Example 175
1-(3-Mercapto-2-methylpropanoyl)-L-proline tert butyl ester
To the cold (5 ) solution of 1.2 g. (lO m~ol.) of 3
mercapto-2-methylpropanoic acid and 1.7 g. ~lO mMol.) of
L-proline tert. butyl ester in 25 ml. dichloromethane
2.26 g. of dicyclohexylcarbodiimide in 5 ml. dlchloromethane
iS added in portions. After 2 hours at room temperature,
5 drops of acetic acid are added, the mixture is filtered
and the filtrate èvaporated to an oily residue. This
residue is take~ up in 20 ml. of petroleum ether-ethyl
acetate (3:1) and applied to a 150 ml. silica gel column
prepared in petroleum ether. The fraction eluted with
3~ petroleum ether-ethyl acetate (1:1) contains the product,
-81-
.
11~18~4 HA135b
1-(3-mercapto-2-methylpropanoyl)-L-proline tert.butyl ester.
This raction (0.6 g.) is dried over P205 in vacuo or
12 hours. Rf 0.6 (Silica gel, Benzene/Acetic acid 3:1),
Rf 0.8 (Silica gel, Methyl-ethylketone/acetic acid/pyridine/
water 14:1:2:1).
Example 176
1-(3-Mercapto-2-methylpropanoyl)-L-proline
By substituting 1-(3-mercapto-2-methylpropanoyl)-L-
proline tert. butyl ester for the l-(3-mercaptopropanoyl-L-
proline tert.butyl ester in the procedure of Example 18C,
1-(3-mercapto-2 methylpropanoyl)-L-proline is obtained.
R~ 0.35 ~Silica gel, Benzene/acetic acid 3:1), Rf 0.5
(Silica gel, Methyl-ethylketone/Acetic acid/Pyridine/Water
14:1:2:1), identical ~o t~ compo~lod of Ex~rplo 34
:
-82-
36~
HA135b
The racemic form of ~he linal product in any of
the foregoing examples is produced by utilizing the ~L-form
of the starting amino acid instead of the L-~orm.
~ imilarly, the D-form of the final products in any
of the foregoing examples is produced ~y utiliziny the D-form
of the starting amino acid instead of the L-form.
Example 177
.
lO00 ta~lets each containing lO0 mg. bf l-(2-
mercaptopropanoyl)-L~proline are produced from the following
lO ingredients:
l-(2-Mercaptopropanoyl)-L-prolinelO0 ~.
Corn starch 50 g.
Gela-tin 7-5 g-
Avicel (microcrystalline ccllulose) 25~ g-
Magnesium stearate ~ 2.5 y.
The l-(2-mercaptopropanoyl)-L-proline and corn
starch are admixed with an aqueous solutlon of the gelatin.
The mixture is dried and ground to a fine powder. The
Avicel and then the magnesium stearate are admixed with the
granulation. This is then compressed in a tablet to ~orm
lO00 tablets each containing lO0 mg. of active ingredient.
xampIe 178
By substituting lO0 y. o~ l-(3-mercapto-2-D-
methylpropanoyl)-L-proline for the l-(2-mercaptopropanoyl)-
L proline in Example 177, 1000 tablets each containing lO0 mg.
of the l-(3-mercapto-2-D-methylpropanoyl-L-proline are
produced.
,
-83-
:; :: : : .
- : . . ...
5164
HA135b
Example_ 179
1000 tablets each co~taining 200 mg. of 1-(2-
mercaptoacetyl)-L-proline are produced from the following
ingredients:
1-(2-Mercaptoacetyl)-L-proline200 g.
Lactose 100 g.
Avicel 150 g.
Corn starch 50 y.
Magnesium stearate 5 g.
The 1-(2-mercaptoacetyl)-~-proline, lac~ose and
Avicel are admixed, then blended with the corn starch.
Magnesium stearate is added. The dry mixture is compressed
in a tablet press to form 1000 505 mg. tablets each containing
200 mg. of active ingredient. The tablets are coated ~ith a
solution of Methocel E 15 (methyl cellulose) including as a
color a lake containing yellow #6.
Example I80 ~ ~
Two piece #1 gelatin capsules each containing 2~0 mg.
of 1-(2-mercaptopropanoyl)-L-proline are filled wlth a mixture
of the following ingredients:
1-(2-Mercaptopropanoyl~-L-proline 250 mg.
Magnesium stearate 7~ mg.
USP lactose 193 mg.
Example 181
An injectable solution is produced as follows:
1-(2-Mercaptopropanoyl)-L-prollne500 mg.
Methyl paraben 5 g.
Propyl paraben -L g~
Sodium chloride 25 g.
Wa-Lcr for injec~io~ 5 1.
-84-
6~
HAl35b
he active substance, preservatives and sodium
chloride are dissolved in 3 li~ers of water for injcction and
then the volume is hrought up to 5 liters. The solution is
filtered through a sterile Eilter and aseptically filled
into presterilized vials which are then closed with pre-
sterilized rubber closures. Each vial contains 5 ml. of
solu~ion in a concentration of lO0 my. of active ingredient
per ml. o solution for injection.
Example 182
By substituting lO0 g. oE l,l'-[dithiobis(2-D-methy~l-
3-propanoyl)~-bis-L-proline or the l-(2-mercaptopropanoyl)-
L-proline in Example 177, lO00 tablets each containing lO0 mg.
of the l,l'-[dithiobisl2-D-methyl-3-propanoyl)]-bis-L
proline are produced.
Each oE the products o~ the examples can be
similarly formulated by substi-tuting it for ~he active
_ngredlent in ~xamples 177, 179, 180 or 181
:
', ;.~'' . '
,
-
-as-
., ~
